Methods of detecting target nucleic acids

ABSTRACT

The present disclosure relates to methods of identifying target nucleic acids by using coded molecules and its analysis by translocation through a nanopore. Generally, coded molecules are subject to a target polynucleotide dependent modification. The modified coded molecule is detected by isolating the modified coded molecules from the unmodified coded molecules prior to analysis through the nanopore or by detecting a change in the signal pattern of the coded molecule when analyzed through the nanopore.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) to applicationSer. No. 60/781,780, filed Mar. 12, 2006, the contents of which areincorporated herein by reference.

2. BACKGROUND

The detection of target nucleic acids has many critical applications inmedicine, forensics, and environmental monitoring. To provideconsistency, speed, and specificity in detecting multiple targetpolynucleotides, various mutiplexing techniques have been developed inwhich detection is carried out in a single reaction. Multiplexingapproaches based on microarrays and microbeads combine powerful nucleicacid amplification strategies with the massive screening capability toprovide highthroughput capacity along with high level of sensitivity,specificity, and consistency.

Micoarrays and microbeads are particularly suited for detecting singlenucleotide polymorphisms, which represents one of the largest sources ofdiversity in the genome of organisms. Some single nucleotide variationsare directly linked to phenotypic traits of interest, such as a diseaseor disease susceptibility. Most single nucleotide polymorphisms,however, are neutral. But because most biological processes involve theinteraction of a multitude of genes, even neutral sequence variationsserve as useful markers in linkage maps for studying phenotypes havingan underlying multigenic basis. The growing number of SNPs, for examplethe SNP database maintained by the National Center for BiotechnologyInformation (NCBI), provide a rich resource for genetic analysis basedon sequence polymorphisms.

Despite the advances of microarray and microbead based nucleic aciddetection systems, these methods still have drawbacks. For example, theneed to amplify the target nucleic acid in many applications representsa disadvantage because of variability in amplification efficiency. Sincemicroarray and microbead techniques typically compare signal intensitybetween samples for determining a positive or negative result,variability in amplification reactions can adversely affect thedetermination of the presence or absence of a particular target nucleicacid in these assay formats. Moreover, microarray and microbead baseddetection typically rely on summing of signals from a population ofprobe-target nucleic acid interactions, which limits the sensitivity ofthe assays.

In view of the foregoing, it is desirable to have alternative techniquesfor detecting nucleic acids, where the detection technique is lesssusceptible to variation in amplification efficiency, displays a highlevel of sensitivity, and is adaptable for multiplexing reactions todetect SNPs.

3. SUMMARY

The present disclosure relates to use of nanopores to detect targetpolynucleotides. The methods involve use of coded molecules having anassociated target probe that specifically recognizes a targetpolynucleotide; modifying the coded molecule with a modifying agent,where the modification is dependent on presence of the targetpolynucleotide; and detecting the modified coded molecule bytranslocating it through a nanopore.

Generally, the method comprises contacting a coded molecule with atarget polynucleotide, wherein the coded molecule comprises one or moreblock polymer regions and a target probe capable of hybridizing to thetarget polynucleotide. The mixture of coded molecule and the targetpolynucleotide is treated with a modifying agent that modifies thetarget probe if the target polynucleotide is hybridized to the targetprobe. In the absence of a hybridized target polynucleotide, themodification of the target probe, and thus modification of the codedmolecule, does not occur efficiently. The coded molecule is thentranslocated through a nanopore and interrogated to detect a signal thatis reflective of the polymer characteristics of the block polymerregion. In some embodiments where there is a modification of the codedmolecule, the modification can alter the signal pattern displayed by acoded molecule such that an altered signal pattern is indicative of thepresence of the target polynucleotide. The detected signal pattern isalso used to identify the specific coded molecule and its associatedtarget probe, and thus the specific target polynucleotide detected. Insome embodiments, the modified coded molecule can be isolated fromunmodified coded molecules based on the target polynucleotide dependentmodification, and the isolated coded molecule translocated through ananopore to detect a signal pattern, which can then be associated to aspecific coded molecule and therefore the specific target polynucleotidedetected in the reaction.

In the present disclosure, the target probes are designed to hybridizeto a target polynucleotide to form polynucleotide structures that can bemodified by various modifying agents. In some embodiments, the targetprobe comprises a 3-prime region or segment that hybridizes to a 5-primeregion of a target polynucleotide such that the hybridized 3-primeregion of the target probe can serve as a primer for elongation by atemplate-dependent polymerase. In some embodiments, extension of the3-prime region of the target probe can alter the signal pattern becauseof the polynucleotide segment added to the coded molecule by thetemplate-dependent polymerase. Because the extension does not occur inthe absence of a template, changes to the signal pattern can occur onlyin the presence of the target polynucleotide. In some embodiments, thetarget polynucleotide is a circular polynucleotide formed by ligation ofan open circle probe, where the open circle probe is efficiently ligatedto form the circular target polynucleotide only in the presence of anucleic acid of interest. Elongation of the target probe by replicationof the circular polynucleotide provides a basis for determining thepresence or absence of circular polynucleotide, and thus the presence orabsence of the nucleic acid of interest.

In some embodiments, the method further comprises hybridizing a ligationprobe to the target polynucleotide, where the ligation probe and thetarget probe hybridize to adjacent regions on the target polypeptidesuch that the hybridized ligation probe and target probe are suitablesubstrates for a ligase. Treatment with a ligase results in ligation ofthe ligation probe to the target probe, thereby structurally modifyingthe coded molecule. A change in the detected signal pattern, as comparedto the signal pattern of an unmodified coded molecule, indicates thepresence of the target polynucleotide. In some embodiments, the ligationprobe can further comprise a signal generating segment, which is apolymer segment that can change the signal pattern of the codedmolecule. In other embodiments, the ligation probe comprises a capturetag, which allows the ligase-modified coded molecule to be isolated fromunmodified coded molecules. In various embodiments, the ligation probeor the target probe can be used to interrogate a site of nucleotidesequence variation.

In some embodiments, the method further comprises hybridizing a FLAPprobe to the target polynucleotide to form a FLAP structure recognizedby a FLAP endonuclease and consequent cleavage of the target probe by aFLAP endonuclease. In these embodiments, the FLAP substrate comprises(a) a target polynucleotide, wherein the target polynucleotide comprisesadjacent first and second regions, (b) a FLAP probe comprising a 3-primesegment that hybridizes to the first region, and (c) a target probecomprising a 5-prime region and a 3-prime region, where the 3-primeregion hybridizes to the second region of the target polynucleotide.Hybridization of the FLAP probe and the target probe to the adjacentfirst and second regions on the target polynucleotide forms a FLAPsubstrate in which the target probe is cleaved by a FLAP endonuclease,thereby resulting in separation of the 5-prime region of the targetprobe from the coded molecule. In some embodiments, a change in thesignal pattern arising from cleaving off of the 5-prime region of thetarget probe indicates the presence of the target polynucleotide. Use oftarget probes having a signal generating segment can assist indistinguishing unmodified coded molecules from FLAP-endonucleasemodified coded molecules. In other embodiments, the target probe canhave a capture tag, which is removed by action of the FLAP endonuclease,thus permitting isolation of FLAP-endonuclease modified coded moleculesfrom unmodified coded molecules. In various embodiments, the FLAP probeor the target probe can be used interrogate a site of polynucleotidepolymorphism to determine the presence or absence of a nucleotidesequence variation.

In some embodiments, the method comprises hybridizing a target probe toa target polynucleotide to form an endonuclease recognition site and acorresponding endonuclease cleavage site. In these embodiments, treatingthe hybridized target polynucleotide and the target probe with anendonuclease that specifically recognizes the recognition site resultsin cleavage of the target probe. In some embodiments, the endonucleaserecognition site can be a sequence-specific endonuclease site while inother embodiments, the endonuclease recognition site can be amismatch-specific endonuclease. Use of target probes having a signalgenerating segment can be used to distinguish unmodified coded moleculesfrom endonuclease-modified coded molecules.

In some embodiments, a target probe is hybridized to the targetpolynucleotide to form a double stranded region in which the targetprobe is made susceptible to an exonuclease. Removal of all or a portionof the target probe alters the structure of the coded molecule, whichcan result in an altered signal pattern indicative of the presence of aspecific target polynucleotide.

The present disclosure further provides multiplexed detection based onthe methods described above. Detection of a plurality of differenttarget polynucleotides can be carried out using subpopulations of codedmolecules, i.e., a plurality of pluralities, where each subpopulationhas a target probe that hybridizes to a specific target polynucleotidedifferent from the target polynucleotide bound by the targets probes ofthe other subpopulations. Each member of a subpopulation of codedmolecules can display a signal pattern that is distinguishable fromcoded molecules of other subpopulations. By detecting the signal patternof a translocated coded molecule, the signal pattern can be associatedto a specific coded molecule subpopulation, and thus the specific targetpolynucleotide detected in the assay.

Interrogation of the coded molecule following treatment with themodifying agent is carried out by translocating the coded moleculethrough a nanopore and detecting the signal pattern associated with thecoded molecule. Various detection strategies are contemplated, includingcurrent blockade, electron tunneling current, and imaging of chargeinduced fields. The detected signal pattern is analyzed and associatedto a specific coded molecule and its corresponding target probe.

The methods herein can be used to detect any target polynucleotide, suchas polynucleotides associated with diseases and other medical conditionsas well as polynucleotides with sequence variations useful forgenotyping applications, such as forensic analysis, environmentalsampling, and evolutionary studies.

Further provided herein are kits for the detection of targetpolynucleotides by the disclosed methods. Kits can contain codedmolecules with target probes directed to various target polynucleotides,modifying agents for the modification of the coded molecules, andnanopore devices for detecting the coded molecules. In addition, thekits can contain various coded molecule standards for obtainingrepresentative signal pattern profiles of unmodified coded molecules andrepresentative signal pattern profiles of modified coded molecules forcomparison of test samples. In various embodiments, the kits can alsocontain instructions on use of kit components and nanopore detectionmethodology, wherein the instructions can be in any medium used fordisseminating such information, including, among others, printed medium,video tape, compact disc, flash memory devices, and computer disc.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is an illustration of a polymerase-mediated extension assay inwhich a coded molecule is modified by a template-dependent polymerasemediated extension of the 3-prime region or segment of the target probehybridized to the target polynucleotide. The segments represented by (-)( - - - ), and ( . . . ) comprise block polymer regions. FIG. 1Bsymbolically illustrates a current blockade signal profile for anunmodified coded molecule and polymerase-modified coded molecule.

FIG. 2A is an illustration of a primer extension assay in which a targetprobe segment of the coded molecule hybridizes to a ligated open circleprobe (OCP). Polymerase-mediated extension of the coded molecule resultsin a coded molecule with an extended 3-prime region or segment that cancomprise multiple copies of the circular template. FIG. 2B symbolicallyillustrates a current blockade signal profile for an unmodified codedmolecule and a polymerase-modified coded molecule.

FIG. 3A is an illustration of a ligation assay in which a target probeof the coded molecule is ligated to a ligation probe. The target probeand the ligation probe are adjacently hybridized to a targetpolynucleotide to juxtapose the terminus of the target probe andligation probe. Treatment with a ligase ligates the ligation probe tothe target probe. In the illustrated embodiment, the ligation probecomprises a signal generating segment, which displays a unique currentblockade signal profile. FIG. 3B symbolically illustrates a currentblockade signal profile for an unmodified coded molecule and a codedmolecule modified by ligation of a ligation probe.

FIG. 4A is an illustration of a FLAP endonuclease assay in which a FLAPprobe and a 3-prime region of the target probe are adjacently hybridizedto a target polynucleotide. The target probe, FLAP probe, and targetpolynucleotide form a FLAP substrate in which the target probe iscleaved by a FLAP endonuclease to release the 5-prime region of thetarget probe. In the illustrated embodiment, the target probe comprisesa signal generating segment that displays a unique signal profile toclearly distinguish a coded molecule with and without the signalgenerating segment. FIG. 4B symbolically illustrates a current blockadesignal profile for an unmodified coded molecule and a coded moleculecleaved by a FLAP endonuclease. FLAP endonuclease modification resultsin release of the 5-prime region of the target probe and loss of theassociated signal generating segment.

FIG. 5A is an illustration of a sequence or mismatch specificendonuclease assay in which the target probe and the targetpolynucleotide hybridize to form a recognition site for thesequence-specific or mismatch specific endonuclease. In the illustratedembodiment, the target probe has attached a signal generating segmentthat generates a unique signal to clearly distinguish a coded moleculewith and without the signal generating segment. FIG. 5B symbolicallyillustrates a current blockade signal profile for an unmodified codedmolecule and a coded molecule modified by a sequence-specific or amismatch specific endonuclease. Cleavage by the endonuclease results inrelease of a portion of the target probe and its associated signalgenerating segment.

5. DETAILED DESCRIPTION

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure. In this disclosure, the use of the singular includes theplural unless specifically stated otherwise. Also, the use of “or” means“and/or” unless stated otherwise. Similarly, “comprise,” “comprises,”“comprising” “include,” “includes,” and “including” are not intended tobe limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

The section headings used herein are for organizational purposes onlyand not to be construed as limiting the subject matter described.

5.2 Definitions

As used throughout the instant application, the following terms shallhave the following meanings:

“Nucleobase” or “Base” means those naturally occurring and syntheticheterocyclic moieties commonly known in the art of nucleic acid orpolynucleotide technology or polyamide or peptide nucleic acidtechnology for generating polymers that can hybridize to polynucleotidesin a sequence-specific manner. Non-limiting examples of suitablenucleobases include: adenine, cytosine, guanine, thymine, uracil,5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitablenucleobases include those nucleobases illustrated in FIGS. 2(A) and 2(B)of Buchardt et al. (WO 92/20702 or WO 92/20703). Nucleobases can belinked to other moieties to form nucleosides, nucleotides, andnucleoside/tide analogs.

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil,thymine, 7-deazaadenine, 7-deazaguanosine, that is linked to theanomeric carbon of a pentose sugar at the 1′ position, such as a ribose,2′-deoxyribose, or a 2′,3′-di-deoxyribose. When the nucleoside base ispurine or 7-deazapurine, the pentose is attached at the 9-position ofthe purine or deazapurine, and when the nucleoside base is pyrimidine,the pentose is attached at the 1-position of the pyrimidine (see, e.g.,Kornberg and Baker, 1992, DNA Replication, 2nd Ed., Freeman. The term“nucleotide” as used herein refers to a phosphate ester of a nucleoside,e.g., a mono-, a di-, or a triphosphate ester, wherein the most commonsite of esterification is the hydroxyl group attached to the C-5position of the pentose. “Nucleotide 5′-triphosphate” refers to anucleotide with a triphosphate ester group at the 5′ position. The term“nucleoside/tide” as used herein refers to a set of compounds includingboth nucleosides and/or nucleotides.

“Nucleobase polymer” or “Nucleobase oligomer” refers to two or morenucleobases that are connected by linkages that permit the resultantnucleobase polymer or oligomer to hybridize to a polynucleotide having acomplementary nucleobase sequence. Nucleobase polymers or oligomersinclude, but are not limited to, poly- and oligonucleotides (e.g., DNAand RNA polymers and oligomers), poly- and oligonucleotide analogs andpoly- and oligonucleotide mimics, such as polyamide or peptide nucleicacids. Nucleobase polymers or oligomers can vary in size from a fewnucleobases, from 2 to 40 nucleobases, to several hundred nucleobases,to several thousand nucleobases, or more.

“Polynucleotides” or “Oligonucleotides” refers to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Exemplary poly- andoligonucleotides include polymers of 2′-deoxyribonucleotides (DNA) andpolymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′-deoxyribonucleotides orcombinations thereof The term nucleic acid encompasses the termspolynucleotide and oligonucleotides and includes single stranded anddouble stranded polymers of nucleotide monomers.

“Polynucleotide analog” or “Oligonucleotide analog” refers to nucleobasepolymers or oligomers in which the nucleobases are connected by a sugarphosphate backbone comprising one or more sugar phosphate analogs.Typical sugar phosphate analogs include, but are not limited to, sugaralkylphosphonates, sugar phosphoramidites, sugar alkyl- or substitutedalkylphosphotriesters, sugar phosphorothioates, sugarphosphorodithioates, sugar phosphates and sugar phosphate analogs inwhich the sugar is other than 2′-deoxyribose or ribose, nucleobasepolymers having positively charged sugar-guanidyl interlinkages such asthose described in U.S. Pat. No. 6,013,785 and U.S. Pat. No. 5,696,253(see also, Dagani, 1995, Chem Eng News 4-5:1153; Dempey et al., 1995, JAm Chem Soc 117:6140-6141). Such positively charged analogues in whichthe sugar is 2′-deoxyribose are referred to as “DNGs,” whereas those inwhich the sugar is ribose are referred to as “RNGs.” Specificallyincluded within the definition of poly- and oligonucleotide analogs arelocked nucleic acids (LNAs; see, e.g., Elayadi et al., 2002,Biochemistry 41:9973-9981; Koshkin et al., 1998, J Am Chem Soc120:13252-3; Koshkin et al., 1998, Tetrahedron Letters, 39:4381-4384;Jumar et al., 1998, Bioorganic Medicinal Chemistry Letters 8:2219-2222;Singh and Wengel, 1998, Chem Commun 12:1247-1248; WO 00/56746; WO02/28875; and WO 01/48190; all of which are incorporated herein byreference in their entireties) and nucleic acids with sugar-phosphatesother than deoxyribose- or ribose-phosphate backbone, for example,hexopyranosyl-phosphate backbones (Eschenmoser, 1999, Science284:2118-2124).

“Polynucleotide mimic” or “Oligonucleotide mimic” refers to a nucleobasepolymer or oligomer in which one or more of the backbone sugar-phosphatelinkages is replaced with a sugar-phosphate analog. Such mimics arecapable of hybridizing to complementary polynucleotides oroligonucleotides, or polynucleotide or oligonucleotide analogs or toother polynucleotide or oligonucleotide mimics, and may includebackbones comprising one or more of the following linkages: positivelycharged polyamide backbone with alkylamine side chains as described inU.S. Pat. No. 5,786,461; U.S. Pat. No. 5,766,855; U.S. Pat. No.5,719,262; U.S. Pat. No. 5,539,082; and WO 98/03542 (see also, Haaima etal., 1996, Angewandte Chemie Int'l Ed. in English 35:1939-1942; Lesnicket al., 1997, Nucleosid Nucleotid 16:1775-1779; D'Costa et al., 1999,Org Lett 1:1513-1516 see also Nielsen, 1999, Curr Opin Biotechnol10:71-75); uncharged polyamide backbones as described in WO 92/20702 andU.S. Pat. No. 5,539,082; uncharged morpholino-phosphoramidate backbonesas described in U.S. Pat. No. 5,698,685, U.S. Pat. No. 5,470,974, U.S.Pat. No. 5,378,841 and U.S. Pat. No. 5,185,144 (see also, Wages et al.,1997, Bio Techniques 23:1116-1121); peptide-based nucleic acid mimicbackbones (see, e.g., U.S. Pat. No. 5,698,685); carbamate backbones(see, e.g., Stirchak and Summerton, 1987, J Org Chem 52:4202); amidebackbones (see, e.g., Lebreton, 1994, Synlett 1994:137); methylhydroxylamine backbones (see, e.g., Vasseur et al., 1992, J Am Chem Soc114:4006); 3′-thioformacetal backbones (see, e.g., Jones et al., 1993, JOrg Chem 58:2983); sulfamate backbones (see, e.g., U.S. Pat. No.5,470,967); and α-threofuranosyl backbones (Schoning et al., Science2901347-1351). All of the preceding publications are incorporated hereinby reference.

“Peptide Nucleic Acid” or “PNA” refers to poly- or oligonucleotidemimics in which the nucleobases are connected by amino linkages(polyamide backbone) such as described in any one or more of U.S. Pat.Nos. 5,539,082; 5,527,675; 5,623,049; 5,714,33; 5,718,262; 5,736,336;5,773,571; 5,766,855; 5,786,461; 5,837,459; 5,891,625; 5,972,610;5,986,053; 6,107,470; 6,451,968; 6,441,130; 6,414,112; and 6,403,763;all disclosures of which are incorporated herein by reference. The term“peptide nucleic acid” or “PNA” shall also apply to any oligomer orpolymer comprising two or more subunits of those polynucleotide mimicsdescribed in the following publications: Lagriffoul et al., 1994, BioorgMed Chem Lett 4: 1081-1082; Petersen et al., 1996, Bioorg Med Chem Lett6:793-796; Diderichsen et al., 1996, Tett. Lett. 37: 475-478; Fujii etal., 1997, Bioorg Med Chem Lett 7:637-627; Jordan et al., 1997, BioorgMed Chem Lett 7:687-690; Krotz et al., 1995, Tett Lett 36:6941-6944;Lagriffoul et al, 1994, Bioorg Med Chem Lett 4:1081-1082; Diederichsen,U., 1997, Bioorg Med Chem Lett 7:1743-1746; Lowe et al., 1997, J ChemSoc Perkin Trans 1:539-546; Lowe et al., 1997, J Chem Soc Perkin Trans11:547-554; Lowe et al., 1997, J Chem Soc Perkin Trans 1:555-560;Howarth et al., 1997, J Org Chem 62:5441-5450; Altmann, K-H et al.,1997, Bioorg Med Chem Lett 7:1119-1122; Diederichsen, U., 1998, BioorgMed Chem Lett 8:165-168; Diederichsen et al., 1998, Angew. Chem. mt.Ed., 37: 302-305; Cantin et al., 1997, Tet Lett 38:4211-4214; Ciapettiet al., 1997, Tetrahedron 53:1167-1176; Lagriffoule et al., 1997, ChemEur J 3:912-919; Kumar et al., 2001, Org Lett 3(9):1269-1272; and thePeptide-Based Nucleic Acid Mimics (PENAMs) disclosed in WO 96/04000.Some examples of PNAs are those in which the nucleobases are attached toan N-(2-aminoethyl)-glycine backbone, i.e., a peptide-like, amide-linkedunit (see, e.g., U.S. Pat. No. 5,719,262; WO 92/20702; and Nielsen etal., 1991, Science 254:1497-1500). All publications are incorporatedherein by reference.

“Chimeric oligonucleotide” or “Chimeric polynucleotide” refers to anucleobase polymer or oligomer comprising a plurality of differentpolynucleotides, polynucleotide analogs and polynucleotide mimics. Forexample, a chimeric polymer may comprise a sequence of DNA linked to asequence of RNA. Other examples of chimeric polymers include a sequenceof DNA linked to a sequence of PNA or a sequence of RNA linked to asequence of PNA.

“Detectable tag” refers to a moiety that, when attached to anothermolecule, e.g., an oligonucleotide, nucleobase polymer, a targetpolynucleotide, renders such molecule detectable using known detectionmethods, e.g., spectroscopic, photochemical, electrochemiluminescent,and electrophoretic methods. A detectable tag may have one or more thanone label, including different types of labels. Exemplary tags include,but are not limited to, fluorophores, radioisotopes, nanoparticles, andquantum dots. Such tags allow direct detection of labeled compounds by asuitable detector, e.g., a fluorometer.

“Capture tag” refers to a member of a binding pair that, when attachedto another molecule, e.g., a nucleotide, oligonucleotide, nucleobasepolymer, a target polynucleotide, allows the isolation of the molecule(i.e., captured) by interaction with the other member of the bindingpair. A capture tag may have one or more than one tag, includingdifferent types of capture tags. Exemplary capture tags include, amongothers, biotin, which can be incorporated into nucleic acids (Langer etal., 1981, Proc Natl Acad Sci USA 78:6633) and captured usingstreptavidin or biotin-specific antibodies; a hapten such as digoxigeninor dinitrophenol (Kerkhof, 1992, Anal Biochem 205:359-364), which can becaptured using a corresponding antibody; a fluorophore to whichantibodies can be generated (e.g., Lucifer yellow, fluorescein, etc.);and a metal binding domain (e.g., His tag), which can be captured usinga suitable metal ligand. In some embodiments, the capture tag cancomprise a specific nucleobase sequence, referred to as a “capturesequence,” which can be captured using a “capture probe” having asequence complementary to the capture sequence.

“Watson/Crick Base-Pairing” refers to a pattern of specific pairs ofnucleobases and analogs that bind together through sequence-specifichydrogen-bonds, e.g., A pairs with T and U, and G pairs with C, commonlyobserved in double stranded nucleic acid.

“Annealing” or “Hybridization” refers to the base-pairing interactionsof one nucleobase polymer with another that results in the formation ofa double-stranded structure, a triplex structure or a quaternarystructure. Annealing or hybridization can occur via Watson-Crickbase-pairing interactions, but may be mediated by other hydrogen-bondinginteractions, such as Hoogsteen or Reverse-Hoogsteen base pairing.

“Deoxynucleotide triphosphates” or “dNTPs” refer to deoxynucleosidetriphosphate precursors, i.e., dATP, dTTP, dGTP, and dCTP, and dUTP.

“Wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term “mutant”refers to a gene or gene product which displays modifications insequence and or functional properties (i.e., altered characteristics)when compared to the wild-type gene or gene product. It is noted thatnaturally-occurring mutants can be isolated; these are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product.

“Sequence variation” as used herein refers to differences in nucleicacid sequence between two nucleic acids. For example, a wild-typestructural gene and a mutant form of this wild-type structural gene mayvary in sequence by the presence of single base substitutions, deletionsand/or insertions of one or more nucleotides. These two forms of thestructural gene are said to vary in sequence from one another. A secondmutant form of the structural gene may exist. This second mutant form issaid to vary in sequence from both the wild-type gene and the firstmutant form of the gene.

5.3 Methods of Detecting Target Polynucleotides

The present disclosure provides methods of detecting targetpolynucleotides by hybridizing coded molecules to the targetpolynucleotides and modifying the coded molecules with a modifyingagent, where the modification is dependent on the presence of ahybridized target polynucleotide. In the embodiments herein, the codedmolecule comprises one or more block polymer regions, thecharacteristics of which can be detected by translocating the codedmolecule through a nanopore and detecting a signal associated with theblock polymer regions. Generally, the detected signal will varydepending on the length and polymer composition of each block polymerregion. The signal pattern or signal profile displayed by a codedmolecule as a whole can be used as a signature for the specific codedmolecule.

In various embodiments, the modification reaction alters the codedmolecule, allowing the coded molecule to be separated from unmodifiedcoded molecules and/or changing the signal pattern of the modified codedmolecule to distinguish it from a signal pattern of an unmodified codedmolecule. Because the coded molecules in various embodiments can be madesingle-stranded following the modification, both biological and solidstate nanopores selective for single-stranded polymers can be used forinterrogating the coded molecule. Multiplex assays for detecting aplurality of different target polynucleotides are made possible by usingcoded molecules with distinguishable signal patterns generated fromdifferent block polymers or different combinations of block polymersused to form the coded molecule. The scope of multiplexing formats arefurther expanded by employing modification reactions that provideadditional signal pattern profiles that assist in distinguishing amodified the coded molecule from an unmodified coded molecule. The largenumbers of target polynucleotides analyzable with the described methodscan allow the detection of any number of target polynucleotides,including the multiplexed detection of nucleotide sequence variationsassociated with various diseases and the detection of sequencevariations that serve as useful genetic markers.

Generally, the method of detecting a target polynucleotide comprises: a)contacting a coded molecule with a target polynucleotide, wherein thecoded molecule comprises one or more block polymers and a target probecapable of hybridizing to the target polynucleotide; and b) modifyingthe coded molecule with a modifying agent, wherein the modification isdependent on the presence of the target polynucleotide hybridized to thetarget probe. Presence or absence of the modification indicates thepresence or absence of the target polynucleotide in a sample.

The term “target polynucleotide” refers to a defined nucleic acidsequence, the presence or absence of which is being detected. A targetpolynucleotide can be any nucleobase sequence, as further defined below,including but not limited to, DNA, RNA, and chimeric polynucleotides.The target polynucleotide can be obtained from any biological (e.g.,cells, viruses, microbes), environmental (e.g., water, soil, air), orforensic sources. Target polynucleotide also includes any polynucleotidegenerated by any synthetic method, including any chemical syntheticprocess and any polynucleotide amplification technique, for example,polymerase chain reaction (PCR), oligonucleotide ligation assay (OLA),ligase chain reaction (LCR. RCA), reverse transcriptase PCR (RT-PCR),invasive cleavage reaction, strand displacement cleavage, rolling circleamplification, and in vitro transcription reaction.

The term “modified” polynucleotide refers to a polynucleotide that isstructurally altered, such as by addition of nucleotides to one end ofthe polynucleotide, cleavage of the polynucleotide, replacement ofnucleotides, and/or conjugation to another polynucleotide. Variousmodifications can be adapted for the disclosed methods, including, amongothers, extension of a target probe on the coded molecule by atemplate-dependent polymerase, ligation of a ligation probe to thetarget probe, and cleavage of the target probe by a nuclease. Each ofthese modifications is dependent on presence of a target polynucleotidehybridized to the target probe of the coded molecule. In the absence ofthe target polynucleotide, the modification reaction does not occur oroccurs inefficiently such that presence or absence of the modificationis indicative of the presence or absence of the target polynucleotide ina sample.

The coded molecules treated with a modifying agent are interrogated bytranslocating the coded molecule through a nanopore and detecting asignal pattern associated with the coded molecule. In some embodiments,prior to analysis through the nanopore, the coded molecule can be madesingle stranded to permit translocation of the coded molecule through ananopore selective for a single-stranded polymer.

The term “translocating” refers to transporting or passing a codedmolecule through a nanopore such that the coded molecule is interrogatedor scanned from one end of the coded molecule to the other end of thecoded molecule. Translocation conditions can be selected so that thecoded molecule translocates through the nanopore in a substantiallylinear manner.

The term “signal pattern” refers to the reproducible signal profileobtained upon interrogation of the coded molecule by translocationthrough a nanopore and detection of detectable properties of the codedmolecule. Signal pattern profile is related to the chemical and physicalproperties of the polymer(s) that form the coded molecule and thedetection method used to interrogate it. Signal characteristics include,among others, the time dependence of the signal, the signal amplitude,and the temporal appearance of a characteristic signal relative to othersignals.

In various embodiments, the detected signal pattern is associated to aspecific coded molecule. The term “associating” refers to decoding thedetected signal pattern and identifying the specific coded moleculerepresented by that signal pattern. Decoding the detected signal patterncan be done by identifying each element of the signal pattern, forexample, the current blockade signal produced by each of the blockpolymer regions, and determining the specific block polymer regions andtheir arrangement on the detected polymer to ascertain the specificcoded molecule detected. Decoding can be also be performed by comparingthe detected signal pattern to a known set of signal patterns for codedmolecules used in the assay to determine the specific coded moleculedetected.

In various embodiments, the presence of a target polynucleotide can bedetermined by identifying the specific coded molecule translocatedthrough the nanopore based on the detected signal pattern, andascertaining any change in the signal pattern indicative of a targetpolynucleotide dependent modification reaction. In some embodiments, thepresence of the target polynucleotide can be determined by isolating themodified coded molecule away from unmodified coded molecules, such asthrough a capture tag that is attached to the coded molecule in atemplate dependent manner, and then associating the detected signalpattern to a specific coded molecule, and thus the target probe andcorresponding target polynucleotide in the sample. In some embodiments,the presence of a target polynucleotide can be determined via acombination of isolating the modified coded molecule via a capture tagthat is attached to the coded molecule in a template dependent mannerand then ascertaining any change in the signal pattern of the isolatedcoded molecule, indicative of a target polynucleotide dependentmodification reaction.

5.3.1 Coded Molecules

In various embodiments, the coded molecules for the methods hereincomprises polymers with one or more defined regions that produces adefined signal when interrogated by translocation through a nanopore.The polymer can be any type of polymer that can be translocated througha nanopore and detected by any detection method. In some embodiments,the coded molecule comprises a nucleobase polymer, with one or moredefined polymer regions that have a detectable property distinguishablefrom the other portions of the coded molecule. The segment can bedistinguished based on, among others, temporal appearance of the signal;signal magnitude; signal duration, signal type (e.g., conductance ortunneling current), or a combination of the forgoing signalcharacteristics.

In some embodiments, the coded molecule comprises a single chain of anucleobase polymer. In these embodiments, the coded molecule can be asingle-stranded polymer of a polynucleotide, such as a single-strandedDNA, single-stranded RNA, or a single-stranded polynucleotide analog ormimic. In some embodiments where the coded molecule comprises apolynucleotide analog or mimic, any number of nucleobase polymers havinga backbone other than sugar phosphate linkages can be used.

Polynucleotide analogs and mimics include those having linkages of sugaralkylphosphonates, sugar phosphoramidites, sugar alkyl- or substitutedalkylphosphotriesters, sugar phosphorothioates, sugarphosphorodithioates, sugar phosphates and sugar phosphate analogs inwhich the sugar is other than 2′-deoxyribose or ribose, positivelycharged analogue “DNGs” and “RNGs”; positively charged polyamidebackbone with alkylamine side chains; uncharged polyamide backbones;uncharged morpholino-phosphoramidate; peptide-based nucleic acid mimicbackbones; carbamate backbones; amide backbones; methylhydroxyl aminebackbones; 3′-thioformacetal backbones; sulfamate backbones andthreofuranosyl backbones. Exemplary single nucleobase polymers include aglycol nucleic acid with an acyclic three carbonpropyleneglycolphosphodiester backbone and α-threofuranosyl backbones(Schoning et al., supra), both of which can undergo Watson and Crickbase pairing interactions (Zhang et al., 2004, J. Amer. Chem. Soc.Epub). Other types of nucleobase polymers will be apparent to theskilled artisan.

The coded molecules can also be chimeric nucleobase polymers, where thesingle-stranded nucleobase polymer comprises a plurality of differentnucleobase polymers, such as different combinations of polynucleotides,polynucleotide analogs and polynucleotide mimics. Non-limiting examplesof a chimeric nucleobase polymers include, among others, single-strandedpolynucleotides comprising a segment of RNA and a segment of DNA, asegment of RNA and a segment of PNA, or a segment of DNA and a segmentof PNA. Other chimeric nucleobase polymers will be apparent to theskilled artisan.

In other embodiments, the coded molecule is polymer comprising a chimeraof a single stranded nucleobase and non-nucleobase polymers, where thenon-nucleobase polymer regions or linkers connect segments ofsingle-stranded nucleobase polymers. Various synthetic polymers can beused to connect polynucleotide segments together to from a linearpolymer chain. Non-limiting examples of such polymers includepolyethylene glycol (PEG), polystyrenes, polyacrylic acids,polyacetamides, polyphosphates, and other polymers that do not formWatson and Crick or Hoogsteen base pairs with a nucleobase polymer. Thesynthetic polymers can be block polymers or block copolymers. Anon-limiting example of a composite coded molecule is a single strandedpolymer formed with a block polymer of polyethylene glycol and a polymerof deoxypolynucleotides, as described in Sanchez-Quesada et al., 2004,Angew Chem Int Ed 43:3063-3067 and Jaschke et al., 1994, Nucleic AcidsRes. 22(22):4810-4817. Other composite polymers of polynucleotides andnon-polynucleotide polymers or linkers are described in, among others,U.S. Patent Application No. 2005/0153926; Greenberg et al., J Org Chem66:7151-7154; and Pon and Yu, 2005, Nucleic Acids Res. 33(6):1940-1948;the disclosures of which are incorporated herein by reference.

In various embodiments, the block polymer region of the coded moleculecomprises any block polymer that has an associated detectable property.The block polymer can be a block polymer of a purine, purine analog,pyrimidine, or pyrimidine analog. For example, exemplary pyrimidineblock polymers include polyC, polyT, and polyU, while exemplary purineblock polymer include polyA, polyG, and 2,6-diaminoadenine. In otherembodiments, the block polymer region can be a block copolymer, such asan alternating copolymer. The alternating copolymer can be alternatingpurines, alternating pyrimidines or alternating purine/pyrimidine. Anexemplary alternating purine is a region of (AG)_(n), where n is thenumber of repeating units, while an exemplary alternating pyrimidine is(CT)_(n). Exemplary block polymer of alternating purine-pyrimidine is(AC)_(n), (AT)_(n), (GC)_(n), or (GT)_(n). In other embodiments, theblock polymer region can be dinucleotide, trinucleotide, ortetranucleotide repeat sequences, e.g., -(AG)_(n)-, -(CT)_(n),-(ATC)_(n)-, -(TTA)_(n)-, -(AGTC)n, etc., that produce a defineddetectable signal.

In the embodiments herein, any number of block polymer regions can beused to generated the coded molecule. Thus, coded molecule can have atleast 1, at least 2, at least 3, at least 5, at least 10, at least 20,at least 30, up to 50 or more block polymer regions. It is to beunderstood that the number of block polymer regions can be readilydefined by those skilled in the art, taking into consideration variousfactors, which include, among others, block polymer length anddetectable characteristics of the block polymer region. In someembodiments, the coded molecule comprises a plurality of block polymerregions, where a plurality refers to two or more.

In some embodiments, the plurality of block polymer regions can comprisea combination of block polymer regions in which the block polymerregions differ in sequence and/or polymer type. For example, in someembodiments, a first block polymer region can be a polypurine G with adeoxyribophosphate backbone while a second block polymer region can be apolypurine G with a peptide nucleic acid backbone. While the two regionsare made of the same nucleobase, the differences in the backbone areexpected to produce two distinguishable signals when interrogatedthrough the nanopore. As will be apparent to the skilled artisan, anynumber of combinations of block polymer regions of a particularnucleobase and a polymer backbone can be used to generate a diversenumber of coded molecules. Exemplary backbones that can be used todistinguish one block polymer region from another include, PNAbackbones, phosphorothioate backbones, deoxyribophosphate backbones, andribophosphate backbones. Backbones of different isomeric forms, such asRp and Sp phosphorothioate oligonucleotides can be used to distinguishthe block polymer regions (see, e.g., Wilk et al., 2000, J Am Chem Soc122:2149).

The length of the block polymer region can be of any length that issufficient to produce a detectable signal, and can vary depending on thedetection method employed and the property of the block polymer regiondesired. In various embodiments, the block polymer region can be atleast about 5 or more monomer units, about 10 or more monomer units,about 25 or more monomer units, about 50 or more monomer units, about100 or more monomer units, about 200 or more monomer units, about 500 ormore monomer units, about 1000 or more monomer units, up to about 2000or more monomer units. It is to be understood that the length of theblock polymer region can be longer as needed to produce a definedsignal.

In some embodiments, two or more of the block polymer regions areseparated by a non-block polymer region. The term “non-block polymerregion” refers to a polymer region that is not made of the samerepeating monomer unit. An exemplary non-block polymer region is apolymer region of a random sequence of nucleobases. Non-block polymerregion can show a change in the signal between the block polymerregions, which allows distinguishing one block polymer region fromanother block polymer region, even if the two block polymer regions havethe same signal characteristics. Non-block polymer regions of differinglengths in the same range as those described above for the block polymerregions can be used.

In the coded molecules herein, the block polymer regions are ordered onthe coded molecule such that detecting the detectable property generatesa defined signal pattern. “Ordered” as used herein refers to a specifiedspatial arrangement of the block polymer regions on the coded molecule.Any number of block polymer regions can be arranged in variouspermutations to generate a large number of different coded molecules.For example, block polymer regions with the same detectable property canbe positioned at different distances from each other to generate codedmolecules in which the variation in time period between signals producedallows differentiating one coded molecule from another coded molecule.In other variations, a set of block polymer regions can be arranged indifferent spatial arrangements on the single stranded coded molecule togenerate a large number of coded molecules based on a limited number ofblock polymer regions.

In some embodiments, the plurality of block polymer regions is orderedon the coded molecule such that the signal pattern obtained is asymmetric signal pattern. A symmetric signal pattern refers to agenerated signal pattern that is substantially identical when the codedmolecule is translocated through the nanopore beginning from either endof the coded molecule. In still other embodiments, the plurality of theblock polymer regions is ordered on the coded molecule such that thesignal pattern obtained is an asymmetric signal pattern. An asymmetricsignal pattern refers to a generated signal pattern that is notsubstantially identical when the coded molecule enters the pore at oneend of the molecule as compared to the generated signal pattern when themolecule enters the pore from the other end. Thus, a signal pattern thatis asymmetrical allows distinguishing the polarity of the coded moleculetranslocated through the nanopore.

In various embodiments herein, the coded molecule further comprises atarget probe having a region that anneals to a target polynucleotide.The term “target probe” refers to a component of the coded moleculehaving a region complementary or substantially complementary to a targetpolynucleotide and which anneals to the target polynucleotide underreaction conditions suitable for the modification reaction. In someembodiments, the target probe is on the same polymer strand as the codedmolecule. An exemplary target probe of this type is one in which thecoded molecule is a deoxyribonucleotide, and a specific sequence of thedeoxyribonucleotide is complementary to the target polynucleotide. Inother embodiments, the target probe is attached to the coded moleculeindirectly, such as through a linker or by hybridization through acomplementary sequence. One exemplary embodiment of indirect attachmentis where a target probe is hybridized to the target polynucleotide,modified by the modifying agent, and then attached to the coded moleculefor analysis via the region of complementarity between the target probeand the coded molecule. This embodiment generates a coded molecule witha double stranded region, which can be interrogated by translocationthrough the nanopore of sufficient dimension for passage of thedouble-stranded segment. Typically, however, the target probe is on thesame single polymer forming the coded molecule such that the codedmolecule can be interrogated via a nanopore that allows for passage of asingle stranded polynucleotide. In some of these embodiments, thenanopore is selective for passage of a single stranded polynucleotide.

In various embodiments, the target probe is modifiable by the variousmodifying agents described herein. Consequently, the region of thetarget probed involved in the modification reaction is typically asugar-phosphate backbone of naturally occurring polynucleotides. Theseregions include, where appropriate, the terminal regions, such as whenthe modifying agent is a template dependent polymerase, and/or internalregions subject to recognition and cleavage by a nuclease. In someembodiments, the entire target probe is a polynucleotide with a sugarphosphate backbone (e.g., deoxyribonucleic acid or ribonucleic acid). Inother embodiments, the target probe can be a chimeric polynucleotide inwhich one region is a polynucleotide analog or polynucleotide mimic. Anexemplary chimeric target probe of this type is a target probe made of apeptide nucleic acid and a polynucleotide, where the polynucleotideregion of the target probe is modified by a template-dependentpolymerase. For instance, the region hybridizing to the targetpolynucleotide can be made of the peptide nucleic acid while theterminal region extended by the polymerase is made of a deoxyribonucleicacid. Other chimeric polynucleotides suitable a target probes will beapparent to the skilled artisan.

In some embodiments, the target probe of the coded molecule can furthercomprise a signal generating segment, as described in the variousembodiments herein. The term “signal-generating segment” refers to apolymer or polynucleotide of any sequence and/or length that provides adistinctive signal to the signal pattern of the coded molecule.Typically, the signal-generating segment does not hybridize to thetarget polynucleotide, although in some embodiments, the signalgenerating segment can have a region complementary to the targetpolynucleotide. The signal generating segment can be from about 10monomer units or longer, 20 monomer units or longer, 50 monomer units orlonger, 100 monomer units or longer, 500 monomer units or longer, up to1000 monomer units. It is to be understood that in some instances, thesignal generating segment can be longer if needed to distinguish amodified coded molecule from an unmodified coded molecule. The sequenceof the signal-generating segment can be any sequence that produces adistinct signal when interrogated through the nanopore. In someembodiments, the signal generating segment comprises block polymers, asdescribed for coded molecules. Thus, in some embodiments, the signalgenerating segment can comprise a second coded molecule.

An illustrative coded molecule with block polymer regions is given inFIG. 1A, which shows a single stranded polymer with a first blockpolymer region 100, a second block polymer region 102, a third blockpolymer region 103, and a target probe segment 104. A non-block polymerregion 101 separates the first and second block polymer regions, andseparates the signals generated by the first and second block polymerregions. A target polynucleotide 105 is hybridized to a complementaryregion of the target probe. A symbolic representation of currentblockade signal pattern associated with the coded molecule is providedin FIG. 1B.

In the methods herein, the coded molecules in its various forms arecontacted with the target polynucleotide under suitable conditions topermit annealing of the complementary regions of the target probe andthe target polynucleotide. The target probe hybridized to the targetprobes is then treated with a modifying agent to modify the targetprobe, thereby altering the structure of the coded molecule. Thefollowing sections describe various modifying agents suitable for targetpolynucleotide dependent modifications of coded molecules.

5.3.2 Modification of Coded Molecules By Target Polynucleotide DependentPrimer Extension

In some embodiments, the modification is extension of a terminus of thetarget probe by the action of a modifying agent, where the extensionreaction is dependent on presence of a target polynucleotide hybridizedto the target probe. Generally, the modification is targetpolynucleotide directed elongation of the 3-prime terminal region of thetarget probe in which the modifying agent is a template-dependentpolymerase. In these embodiments, the target probe is designed tohybridize to a portion of the target polynucleotide such that thehybridized target polynucleotide has an unhybridized segment that cansupport extension of the target probe. For example, the target probe canbe designed to have 3-prime terminal sequences that hybridize to a5-prime region of the target polynucleotide, where the remaining 3-primeportion of the target polynucleotide remains unpaired and serves as atemplate for extension of the target probe.

The term “elongation” refers to extension of a polynucleotide hybridizedto another polynucleotide, and typically occurs by extension of thepolynucleotide by incorporation of nucleotide triphosphate precursors bya template-dependent polymerase enzyme. The 3-prime terminal nucleotideof the target probe must be properly base-paired to the complementarynucleotide on the target polynucleotide to be elongated by thepolymerase. Thus, in some embodiments, the 3-prime terminal nucleotideof the target probe can be used to interrogate a site of sequencevariation in the target polynucleotide, such as a nucleotidepolymorphism.

In various embodiments, the elongation reaction is carried out in thepresence of one or more nucleotide triphosphates suitable as substratesfor the template dependent polymerase. In some embodiments, theelongation reaction is carried out in presence of all four nucleotidetriphosphates to maximize the elongation reaction and replicate thestrand complementary to the unpaired portion of the targetpolynucleotide.

In some embodiments, the nucleotide triphosphates can be labeled with adetectable tag, such as a fluorophore, hapten, quantum dot,electron-transfer moiety, or a bulky adduct, and detected by the methodsdescribed in U.S. provisional application No. 60/736,960, filed Nov. 14,2005, incorporated herein by reference. As disclosed therein, the labelcan also be detected by their effect on current blockade, electrontunneling current, or charge induced field effects. Detection of labeledsegments can be enhanced by having all four nucleotide triphosphateslabeled with a common detectable tag.

In some embodiments, the nucleotide triphosphates are labeled with acapture tag, which can be used to isolate the modified coded moleculefrom unmodified coded molecules. Exemplary capture tags include, amongothers, biotin, (Langer et al., 1981, Proc Natl Acad Sci USA 78:6633)which can be captured using streptavidin or biotin-specific antibodies;a hapten such as digoxigenin (Kerkhof, 1992, Anal Biochem 205:359-364),which can be captured using an anti-digoxigenin antibody; and afluorophore (e.g., Lucifer yellow, fluorosccinc), which can be capturedalso with a corresponding antibody. In some embodiments, the capture tagcan comprise a specific sequence incorporated into the targetpolynucleotide and which is attached to the coded molecule by thetemplate dependent polymerase. In other embodiments, the capturesequence can be incorporated into a target polynucleotide by ligation ofoligonucleotides or use of primers containing the capture sequence in anamplification reaction. Other capture tags that can be substrates forthe polymerase will be apparent to a skilled artisan. The ability toisolate coded molecules modified with a capture tag provides a method ofdetermining the presence or absence of a target polynucleotide sinceonly those coded molecules having the capture tag will have beenmodified in a target polynucleotide dependent manner. Coded moleculesmodified with a capture tag can be isolated from unmodified codedmolecules and analyzed by translocated through a nanopore.

In some embodiments, the target probe of the coded molecule hybridizesto a region of the target polynucleotide immediately adjacent to thenucleotide base to be identified. In these embodiments, the nucleotidebase to be interrogated is the first unpaired base in the targetpolynucleotide (i.e., template) immediately downstream of the 3-primeterminus of the target probe. Enzymatic extension of the target probe byone nucleotide, catalyzed, for example, by a polymerase, thus depends oncorrect base pairing of the added nucleotide to the nucleotide base tobe identified. In some embodiments, the hybridized target probe can becontacted with a polymerase in presence of four terminators nucleotides(e.g., dideoxynucleotides), each terminator being labeled with adifferent detectable tag or the same or different capture tags. Theduplex of the target probe and the target polynucleotide is contactedwith the polymerase under conditions permitting base pairing of acomplementary terminator nucleotide so as to incorporate the terminatorat the 3-primer terminus of the target probe. Use of chain terminatingnucleotides for interrogating single nucleotide positions on a targetpolynucleotide is described in U.S. Pat. No. 5,88,819, the disclosure ofwhich is incorporated herein by reference.

Various template-dependent polymerases capable of extending the targetprobe hybridized to the target polynucleotide can be used as themodifying agent. These include, among others, DNA polymerases andreverse transcriptases. The polymerase must be primer and templatedependent. Exemplary polymerases include, among others, E. coli DNApolymerase I, “Klenow fragment” of DNA polymerase I, T4 DNA polymerase,T7 DNA polymerase (e.g., Sequenase®), T. aquaticus DNA polymerase, andretroviral reverse transcriptase (e.g., MMLV). The choice of a templatedependent polymerase and selection of the conditions for efficienthybridization and elongation are well within the skill of those in theart. For example, if the 3-prime terminal nucleotide of the target probeis used to interrogate a site of sequence variation in the targetpolynucleotide, a polymerase substantially lacking in a proofreading3-prime to 5-prime exonuclease can be used to minimize removal of theunpaired nucleotide by the exonuclease. In those embodiments in whichthe target polynucleotide is RNA, a reverse transcriptase can be used toextend the 3-terminal nucleotide of the target probe of the codedmolecule.

An illustration of the polymerase mediated extension assay is given inFIG. 1A and FIG. 1B. The illustrated coded molecule is hybridized to thetarget polynucleotide such that the 3-prime region of the targetpolynucleotide 105 remains unpaired. The hybridized 3-terminal region ofthe target probe is then elongated by a template-dependent polymerase byincorporation of nucleotide triphosphate precursors onto the targetprobe 106. After rendering the modified coded molecule single stranded,the coded molecule is translocated through a nanopore and scanned todetect its associated signal pattern. A symbolic representation ofcurrent blockade signal patterns for an unmodified and modified codedmolecules are given in FIG. 1B. Elongation of the coded molecules allowsthe modified coded molecule to be distinguished from the unmodifiedcoded molecule. Further sensitivity in the reaction can be achieved byuse of capture tags in the incorporated nucleotides, which allowsisolation of modified coded molecules from unmodified coded molecules.

5.3.3 Modification of a Coded Molecule By Rolling Circle Replication

In some embodiments, the modification used in the methods herein is apolymerase mediated extension reaction in which the targetpolynucleotide is a sequence on a closed circular nucleic acid to whichthe target probe of the coded molecule hybridizes. Activity of apolymerase extends the target probe, generating tandem copies of thecircular nucleic acid attached to the target probe. This form of primerextension is typically referred to as rolling circle replication and isdescribed in U.S. Pat. No. 6,977,153; 6,858,412; 6,797,474; 6,783,943;6,221,603; and 6,210,884, the disclosures of which are incorporatedherein by reference. The closed circular target polynucleotide isgenerated by ligating an open circle probe (OCP) to which hybridizes anucleic acid sequence of interest. The OCP is a linear nucleic acid thathas a 5-prime phosphate at one end and a 3-prime hydroxyl at the otherend such that the two ends are capable of being ligated by a ligase. Twoterminal portions present on the OPC have sequences complementary to anucleic acid sequence of interest. A first terminal portion comprisessequences at the 5-prime phosphate terminal region while the secondterminal portion comprises sequences at 3-prime hydroxyl terminalregion. The first and second terminal portions hybridize to adjacentsegments on the nucleic acid of interest, thereby by forming adjacent(i.e., abutted) 5-prime and 3-prime terminal nucleotides of the OPC thatserve as substrates for a ligase. In the absence of the specific nucleicacid of interest, ligation of the OCP does not take place.

The open circle further comprises a primer complement portion to whichthe target probe on the coded molecule hybridizes. Typically, a singleprimer complement portion is present on the open circle probe, whichallows rolling circle replication to initiate at one site. The primercomplement portion can be of any length to support hybridization of thetarget probe of the coded molecule to the circular target polynucleotideand can be located any where within the open circle probe. There is nolimitation on the sequence of the primer complement portion as long ithas regions complementary to target probe.

In some embodiments, the open circle probe comprises a capture sequence,which comprises sequences complementary to a capture probe. When theligated circular probe becomes replicated as part of the coded molecule,the modified coded molecule can be isolated through use of a captureprobe that is complementary to the capture tag portion. The open circleprobe may have one or more capture tag portions, which may be the samesequence or different sequences. As discussed above, in otherembodiments, nucleotide triphosphate precursors labeled with capturetags can also be used to isolate coded molecules having tandem copies ofthe circular target polynucleotide.

In still other embodiments, the OCP can have additional sequences togenerate distinctive signal patterns. Thus the OCP can serve as a signalgenerating segment or another coded molecule when replicated. In someembodiments, the additional sequences can comprise one or more blockpolymer regions, which in combination, produce a signal pattern in themanner of a coded molecule. OCPs that produce distinctive signalpatterns when interrogated through a nanopore can be used to enhance themultiplexing capabilities of the coded molecules.

Generally, the rolling circle reaction is carried out by hybridizing anucleic acid of interest to the open circle probe, optionally fillingany gaps with a polymerase and nucleotide triphosphates (or by use of agap oligonucleotide), and ligating the open circle probe to generate aclosed circular probe. A coded molecule having a target probe withsequence complementary to the primer complementary portion is hybridizedto the closed circle probe, and a polymerase added to extend the 3-primeterminal region of the target probe, where the closed circular probeacts as a template for the polymerase. The product formed is a codedmolecule with tandem sequences of the closed circular probe attached tothe target probe. Because the product of rolling circle replication issingle-stranded, the modified coded molecules can be translocateddirectly through a nanopore selective for a single-stranded polymer.Steps for rolling circle modification of a coded molecule typically cancomprise mixing an OCP with a nucleic acid of interest and incubatingthe sample mixture under conditions promoting hybridization between theopen circle probe and the nucleic acid of interest; mixing a ligase withthe OCP-nucleic acid mixture and incubating under conditions promotingligation of the open circle probe; mixing a coded molecule with theligation mixture and incubating under conditions that promotehybridization between the target probe and complementary sequence on theligated open circle probe; and adding a template-dependent polymeraseand incubating under conditions promoting replication of the circulartarget polynucleotide.

DNA polymerases useful in the rolling circle replication will typicallybe capable of displacing the strand complementary to the templatestrand, have low or no 5-prime to 3-prime exonuclease activity, and havehighly processive characteristics. Strand displacement activity isdesirable for synthesizing multiple tandem copies of the ligated OCPwhile low or absent 5-prime to 3-prime exonuclease activity minimizesdestruction of the synthesized strand. Suitable DNA polymerases forrolling circle synthesis, include, among others, bacteriophage φ29 DNApolymerase (U.S. Pat. No. 5,198,543 and U.S. Pat. No. 5,001,050), phageM2 DNA polymerase (Matsumoto et al., 1989, Gene 84:247), phage φPRD1 DNApolymerase (Jung et al., 1987, Proc Natl Acad Sci USA 84:8287), VENT™DNA polymerase (Kong et al., 1993, J Biol Chem 268:1965-1975), Klenowfragment of DNA polymerase I (Jacobsen et al., 1974, Eur J Biochem45:623-627), T5 DNA polymerase (Chatterjee et al., 1991, Gene 97:13-19),PRD1 DNA polymerase (Zhu and Ito, 1994, Biochim Biophys Acta1219:267-276), and T4 DNA polymerase holoenzyme (Kaboord and Benkovic,1995, Curr Biol 5:149-157).

In some embodiments, hybridization of the OCP to the nucleic acid ofinterest leaves a gap between the two terminal portions of the OCP. Thelength of the gap can be from 1 or more nucleotides, 5 or morenucleotides, 10 or more nucleotides, 50 or more nucleotides, or 100 ormore nucleotides. Gaps that are present when the nucleic acid ofinterest hybridizes to the open circle probe can be filled with apolymerase in presence of nucleotide triphosphates to generate a productsuitable for ligation. Polymerases suitable for filling a gap can be anypolymerase capable of acting on the gap, but will generally have low orminimal strand displacement activity to limit displacement of thehybridized portion of the target probe. Suitable gap-filling polymerasesinclude, among others, T7 DNA polymerase (Studier et al., 1990, MethodsEnzymol 185:60-89), T4 DNA polymerase (Kunkel et al., 1987, MethodsEnzymol 154:367-382), Thermus flavus DNA polymerase (MBR, Milwaukee,Wis.), and Stoffel fragment of Taq DNA polymerase (Lawyer et al., 1993,PCR Methods Appl 2(4):275-287; and King et al., 1994, J Biol Chem269(18):13061-13064). In some embodiments, the gap is filled by use ofgap filling oligonucleotide, which hybridizes to the nucleic acid ofinterest in the gap space such that the termini of the gapoligonucleotide is adjacent to the 5-prime terminus and 3-prime terminusof the OPC.

In some embodiments, strand displacement for rolling circle replicationcan be facilitated through the use of strand displacement factors, whichallow use of polymerases lacking strand displacement activity. Exemplarystrand displacement factors useful for rolling circle modification of acoded molecule include BMRF1 polymerase accessory subunit, adenovirusDNA-binding protein, herpes simplex viral protein ICP8, single-strandedDNA binding proteins, and helicase enzymes.

An illustrative embodiment of a rolling circle based modification isgiven in FIG. 2A. The components of the coded molecule are identical tothat illustrated in FIG. 1A. The target polynucleotide 200 is a closedcircular polynucleotide generated by ligation of an OCP hybridized to anucleic acid of interest. The target probe is complementary to a primercomplementary region 201 of the circular target polynucleotide. Treatingthe hybridized complex with a template dependent polymerase results inreplication of tandem copies 201 of the closed circular targetpolynucleotide. A corresponding symbolic representation of currentblockade signal patterns of the unmodified and modified coded moleculesare presented in FIG. 2B. In the illustration, the tandem copies areexpected to produce a repeating signal pattern that follows the signalpattern associated with the block polymer regions of the coded molecule.

5.3.4 Modification of Coded Molecules by Target Polynucleotide DirectedLigation

In some embodiments, the modification is target polynucleotide dependentligation of a ligation probe to the target probe. In these embodiments,the modifying agent is a ligase capable of ligating two adjacentlyhybridized polynucleotides. The term “ligation probe” refers to apolynucleotide capable of hybridizing to a first region of the targetpolynucleotide adjacent to a second region, where the second regionhybridizes to the target probe of the coded molecule. “Adjacent” refersto abutting sequences of the target polynucleotide such that twopolynucleotides hybridized to adjacent regions forms a complex in whicha terminus of one polynucleotide and the terminus of the otherpolynucleotide are adjacent to one another (i.e., no nucleotide gapexists between the two termini). In the embodiments herein,hybridization of the target probe and the ligation probe to the adjacentfirst and second regions on the target polynucleotide forms a complexthat can be ligated by a ligase if suitable 5-prime and 3-prime terminalstructures are present (e.g., 5-prime phosphate and 3-prime hydroxyl).

In some embodiments, the ligation probe and the target probe canhydridize to the target polynucleotide to leave a gap between thehybridized ligation probe and target probe. As noted above, such gapscan be filled by use of a suitable template dependent polymerase inpresence of nucleotide triphosphate precursors suitable as substratesfor the polymerase. Polymerase can extend the 3-prime hydroxyl terminusof one of the polynucleotides hybridized to the target polynucleotide tofill in the gap, thereby generating a ligation probe and target probeadjacent to one another on the target polynucleotide. Alternatively, agap oligonucleotide, as discussed above, can be used to fill in the gap.The use of a combination of ligation probe and gap oligonucleotide (orpolymerase) can be used to interrogate the presence or absence ofparticular sequences in the gap, thereby providing another basis fordistinguishing the presence or absence of a target polynucleotide.

In some embodiments, the length and/or sequence of the ligation probe ischosen to distinguish the signal pattern of a coded molecule modified byligation of the ligation probe from the signal pattern of an unmodifiedcoded molecule. In some embodiments, the portion of the ligation probecomplementary to the target polynucleotide can be made sufficiently longto produce a signal pattern that distinguishes the ligation probemodified coded molecule from an unmodified coded molecule. In variousembodiments, the ligation probe can be from about 10 nucleotides orlonger, about 20 nucleotides or longer, about 50 nucleotides or longer,about 100 nucleotides or longer, about 200 nucleotides or longer, up toabout 500 nucleotides or more as necessary. It is well within theknowledge of the skilled artisan to determined the length of ligationprobe suitable for the purposes herein.

In some embodiments, the ligation probe, in addition to a regioncomplementary to the target probe, has a second region that functions asa signal generating segment. As discussed above, the signal-generatingsegment can be any polymer or polynucleotide of any sequence and lengththat distinguishes the signal pattern of the modified coded moleculefrom the signal pattern of an unmodified coded molecule.

In other embodiments, the ligation probe comprises a label, which may bea detectable tag and/or capture tag. Capture tags can be used to isolatethe coded molecule modified by the modifying agent before being analyzedin the nanopore. Detectable tags can be used to detect the modifiedcoded molecule. As discussed herein, detectable tags that can be used,include, among others, fluorophores, nanoparticles, quantum dots, stericmodifiers, and electron transfer labels. Detection of the detection tagscan use techniques described in U.S. provisional application No.60/736,960, filed Nov. 14, 2006, incorporated herein by reference.

In various embodiments, the terminal nucleotide of either the targetprobe or the ligation probe can be used to interrogate a site ofnucleotide variation in target molecules based on the inability ofligases to ligate two adjacent polynucleotides in which at least one ofthe abutting terminal nucleotides is not complementary to thecorresponding nucleotide on the target polynucleotide. In someembodiments, the target probe interrogates a site of nucleotidepolymorphism on the target polynucleotide. The terminal nucleotide forinterrogating the nucleotide on the target polynucleotide can be a5-prime or 3-prime terminal nucleotide of the target probe. When theinterrogating nucleotide is the 5′-terminal nucleotide of the targetprobe, the ligation probe is designed to hybridize to the targetpolynucleotide such that the 3-prime terminus of the ligation probe isadjacent to the 5-prime terminus of the target probe. When theinterrogating nucleotide is the 3′-terminal nucleotide of the targetprobe, the ligation probe is designed to hybridize to the targetpolynucleotide such that 5-prime terminus of the ligation probe isadjacent to the 5-prime terminus of the target probe. In either case,non-complementarity (i.e., a mismatch) between the nucleotide on thetarget polynucleotide and the interrogating terminal nucleotide of thetarget probe inhibits ligation of the ligation probe to the targetprobe.

In other embodiments, the terminal nucleotide of the ligation probe,rather than the target probe, interrogates a site of nucleotidevariation on the target polynucleotide. In these embodiments, theinterrogating nucleotide can be a 5-prime or 3-prime terminal nucleotideof the ligation probe. When the interrogating nucleotide is the 5-primeterminal nucleotide of the ligation probe, the target probe is designedto hybridize to the target polynucleotide such that the 3-prime terminusof the target probe is adjacent to the 5-prime terminus of the ligationprobe. When the interrogating nucleotide is the 3-prime terminalnucleotide of the ligation probe, the target probe is designed tohybridize to the target polynucleotide such that the 5-prime terminus ofthe target probe is adjacent to the 3-prime terminus of the ligationprobe. In either case, non-complementarity of the opposing nucleotide onthe target polynucleotide and the interrogating terminal nucleotide ofthe ligation probe can inhibit the ligation of the ligation probe to thetarget probe.

In the forgoing embodiments, the modifying agent for the ligationreaction is a ligase. “Ligase” useful for the purposes herein refers tomolecules that covalently link polynucleotides adjacently hybridized tothe target polynucleotide but that fails or is otherwise inefficient inligating free ends of a single-stranded polynucleotide. Ligases can bechemical or enzymatic. Enzymatic ligases include, among others, ATPdependent DNA ligases, NADPH dependent DNA ligases, and RNA ligases.Exemplary ligases include, among others, T4 DNA ligase, E. coli. DNAligase (Panasnko et al., 1978, J Biol Chem. 253:4590-4592), AMPLIGASE™.(Kalin et al., 1992, Mutat Res 283(2):119-123); Winn-Deen et al., 1993,Mol Cell Probes 7(3):179-186), Thermus aquaticus DNA ligase (Barany,1991, Proc Natl Acad Sci USA 88: 189-193), Thermus thermophilus DNAligase, Thermus scotoductus DNA ligase, and Rhodothermus marinus DNAligase (Thorbjarnardottir et al., 1995, Gene 151:177-180). T4 DNA ligaseis suited for ligations involving RNA due to its ability to ligate DNAends involved in DNA:RNA hybrids. DNA ligase and RNA ligase ligates DNAstrands hybridized to a RNA strand (see, e.g., U.S. Pat. No. 6,368,801).T4 RNA ligase joins a 3-prime hydroxyl terminated RNA to a 5-primephosphate terminated RNA (Silber et al., 1972, Proc Natl Acad Sci USA69:3009). RNA molecules hybridized to RNA or DNA strands can be ligatedby T4 RNA ligase.

Various chemical ligases can use reactive groups to join two adjacentlyhybridized polynucleotides. Chemical ligation is described in Naylor andGilham, 1966, Biochemistry 5:2722-2728; Sokolova et al, 1988, FEBS Lett232:153-155; Shabarova, 1988, Biochimie 70:1323-1334; Chu, 1988, NucleicAcids Res. 16:3671-3691; Luebke and Dervin, 1991, J Am Chem Soc113:7447-7448; Luebke and Dervan, 1992, Nucleic Acids Res. 20:3005-3009;Prakash and Kool, 1992, J Am Chem Soc 114:3523-3527; Gryaznov andLetsinger, 1993, J Am Chem Soc 115:3808-3809; Gryaznov and Letsinger,1993, Nucleic Acids Res. 114:9197-9198; and U.S. Pat. No. 5,681,943.

An illustrative ligation assay is provided in FIG. 3A. The codedmolecule is essentially the same as that described for FIG. 1A. Thetarget polynucleotide 301 comprises adjacent first and second regionswhile the ligation probe comprises a 5-prime region 300 that hybridizesto the first region of the target polynucleotide. The target probecomprises a 3-prime region 303 that hybridizes to the second region ofthe target polynucleotide such that the 5-prime terminus of the ligationprobe and the 3-prime terminus of the target probe are adjacent when theligation probe and the target probe are annealed to the targetpolynucleotide. The ligation probe can further comprise a signalgenerating segment 305. Upon treatment of the mixture with ligase, theligation probe is joined to the target probe. The modification resultsin a coded molecule with a signal pattern distinguishable from thesignal pattern of an unmodified coded molecule by the presence ofsignals contributed by the ligation probe and the signal generatingsegment. FIG. 3B symbolically illustrates a current blockade signalpattern for a coded molecule modified by ligation of a ligation probeand its corresponding unmodified coded molecule.

In some embodiments, detecting a plurality of different targetpolynucleotides can use coded molecules that have distinguishable signalpatterns, as further described below. In these embodiments, each of thedifferent coded molecules is associated with a target probe thathybridizes to a specific target polynucleotide. Upon interrogating thecoded molecule by translocation through a nanopore, the detected signalpattern is decoded and associated to a specific coded molecule and itstarget probe. In some embodiments, the presence of a targetpolynucleotide is determined by isolating modified coded molecules fromunmodified molecules through use of captures tags on the ligationprobes. In other embodiments, a change in the detected signal pattern ascompared to a signal pattern of an unmodified coded molecule can be usedas the basis for determining the presence of the target polynucleotide.As will be apparent to the skilled artisan, both approaches can be usedto increase the sensitivity of detecting a target polynucleotide.

In other embodiments, the same coded molecules can be used but incombination with different ligation probes, where each differentligation probe changes the signal pattern of the coded molecule uniquelyto allow a coded molecule modified with one ligation probe to bedistinguished from a coded molecule modified with another ligationprobe. In some embodiments, the coded molecules can hybridize to thesame sequence on different target polynucleotides while the ligationprobes hybridize to sequences that vary between the different targetpolynucleotides. In one exemplification, a first ligation probe cancomprise a first signal generating segment and a second ligation probecomprise a second signal generating segment, where the first and secondsignal generating segment alters the signal pattern of a coded moleculedifferently (i.e., generates differing first signal pattern and secondsignal pattern). Detection of both modified signal patterns indicatesthe presence of first and second target polynucleotides in a sample,while detection of only one of the modified signal pattern indicatespresence of only one of the target polynucleotides. Multiplex detectionof a plurality of different target polynucleotides can use a pluralityof ligation probes in which each ligation probe alters the signalpattern of a coded molecule differently and each ligation probehybridizes to a different target polynucleotide.

5.3.5 Modification of Coded Molecules by FLAP Endonucleases

In some embodiments for detecting a target polynucleotide, the targetprobes are designed to be substrates for a FLAP endonuclease. FLAPendonuclease based assays are described in U.S. Pat. Nos. 5,846,717;5,888,780; 5,985,557; 5,994,069; 6,001,567; 6,090,543; and 6,348,314,the disclosures of which are incorporated herein by reference. In theseembodiments, the target probe forms part of a FLAP structure that is asubstrate for a FLAP endonuclease (see, e.g., Harrington and Lieber,1995, J Biol Chem 270(9):4503-4508). The term “FLAP structure” generallyrefers to a structure comprising (a) a target polynucleotide, whereinthe target polynucleotide has adjacent first and second regions, (b) a5-prime polynucleotide probe comprising a 3-prime region and a 5-primeregion located immediately 5-prime to the 3-prime region, wherein the3-prime region is specifically hybridized to the first region of thetarget polynucleotide, and (c) a 3-prime polynucleotide probe comprisinga 5-prime region specifically hybridized to the second region of thetarget polynucleotide such that the 3-region of the 5′-primepolynucleotide probe and the 5-prime region of the 3-primepolynucleotide probe are specifically hybridized adjacently to the firstand second regions of the target polynucleotide. The 5-prime region ofthe 5-prime polynucleotide probe is an unpaired region, either throughstrand displacement arising from hybridization of the 3-primepolynucleotide probe, the non-complementarity of the 5-prime region tothe target polynucleotide, and/or lower T_(m) of the 5′-prime region forthe target polynucleotide as compared to the 5-prime region of the3-prime polynucleotide probe. In some embodiments, the 5-prime region ofthe 5-prime polynucleotide probe is non-complementary to the targetpolynucleotide.

In the various embodiments herein, the target probe of the codedmolecule functions as the 5-prime polynucleotide probe while a FLAPprobe acts as the 3-prime polynucleotide probe. The 5-prime region ofthe target probe is cleaved off by a FLAP endonuclease or other relatedcleavase enzymes. Thus, for various embodiments based on FLAPstructures, the method of detecting a target polynucleotide comprises(a) contacting a target polynucleotide with a target probe having a5-prime region and a 3-prime region, wherein the target polynucleotidecomprises adjacent first and second regions, and wherein the 3-primeregion of the target probe is capable of hybridizing to the secondregion of the target polynucleotide, (b) contacting the targetpolynucleotide with an FLAP probe, wherein the FLAP probe comprises a5-prime segment capable of hybridizing to the first region of a targetpolynucleotide such that the 5-prime segment of the FLAP probe and the3-prime region of the target probe are adjacently hybridized to thetarget polynucleotide to form a FLAP substrate, and c) treating with aFLAP endonuclease, wherein the FLAP endonuclease is capable ofrecognizing the FLAP substrate and cleaves off the 5-prime region of thetarget probe. Cleavage of the 5-prime region of the target probeseparates a portion of the target probe away from the coded molecule,and thereby provide a basis for altering the signal pattern of themodified coded molecule.

In some embodiments, the FLAP structure is a single FLAP structure. A“single FLAP structure” refers to a FLAP structure having a singleunpaired region formed by the 5-prime region of the 5-primepolynucleotide probe when hybridized to the target polynucleotide. The3-prime polynucleotide probe in the single FLAP structure has its5-prime region specifically hybridized to the first region of the targetpolynucleotide such that there is no unpaired region on the 3-primepolynucleotide probe overlapping with the 5-prime unpaired region of the5-prime polynucleotide probe.

In other embodiments, the FLAP structure is a double FLAP structure(Harrington and Lieber, supra). A “double FLAP structure” refers to aFLAP structure in which the 3-prime polynucleotide probe comprises a3-prime region located immediately 3-prime to a 5-prime region, whereinthe 3-prime region of the 3-prime polynucleotide probe is unpaired andoverlaps with the unpaired 5-prime region of the 5-prime polynucleotideprobe in the FLAP structure. Thus, the double FLAP structure has twounpaired regions, one formed by hybridization of the 5-primepolynucleotide probe and another formed by hybridization of the 3-primepolynucleotide probe, where the two unpaired regions in the double FLAPstructures overlap with one another. These double FLAP structures areshown to serve as more efficient substrates for FEN endonucleases ascompared to the single FLAP structures, even when the unpaired portionon the 3-prime polynucleotide probe is a single nucleotide (Harringtonand Lieber, supra; Kaiser et al., 1999, J Biol Chem. 274:21387-21394).

In some embodiments, the double-stranded FLAP structure comprises a FLAPprobe having a single unpaired nucleotide in the 3-prime region, suchthat a single unpaired nucleotide overlaps with the 5-prime unpairedregion of the 5-prime polynucleotide probe. Besides being efficientsubstrates for a FLAP endonuclease, these double FLAP structures, whencleaved by a FLAP endonuclease, typically results in a structure inwhich the terminus of the FLAP probe is adjacent to the cleaved terminiof the target probe, and therefore ligatable by a ligase. Consequently,the methods based on these FLAP structures can further comprise treatingwith a ligase subsequent to treatment with the FLAP endonuclease. Aswill be apparent from the descriptions herein, the use of a signalgenerating segment on the FLAP probe can provide additional signals thatcan be used to distinguish a FLAP modified coded molecule from anunmodified coded molecule when the FLAP probe is ligated to the cleavedtarget probe.

It is to be understood that the FLAP structures are not limited to theembodiments above. In some embodiments, the FLAP structure is formed byuse of a target probe comprising a first, second, third, and fourthregion, wherein the first region is located 3-prime and the fourthregion is located 5-prime relative to each other. The second and thirdregions are located between the first and fourth regions and arecomplementary to each other such that they hybridize to form a hairpintype structure. The first region is adjacent to the second region andhybridizes to a target polynucleotide such that the targetpolynucleotide has its 3-prime terminus adjacent to the third region ofthe target probe to form a FLAP substrate. The fourth region forms the5-prime unpaired portion in the FLAP substrate, which is cleaved off bya FLAP endonuclease. As noted above, in some embodiments, a 3-primeregion of the target polynucleotide can have an unpaired region to forma double FLAP structure.

In the FLAP substrates based on a hairpin type structure, the targetpolynucleotide can be any polynucleotide being detected, including a5-prime polynucleotide fragment released from another FLAP substrate.Use of two FLAP reactions in which the first FLAP cleavage detects anucleic acid of interest and a second FLAP cleavage detects the cleavageproduct of the first FLAP cleavage reaction can increase the sensitivityof the assay since multiple cycles of hybridization and cleavage can beused to amplify the number of cleaved 5-prime polynucleotide regionsavailable for forming the second FLAP substrate. Such biplex FLAPreactions are described in Lyamichev et al., 1999, Nat. Biotechnol.17:292-296 and Hall et al., 2000, Proc Natl Acad Sci USA 97:8272-8277,the disclosures of which are incorporated herein by reference.

In the embodiments herein, various FLAP endonucleases can be used tocleave the FLAP substrates formed with the target probe, the targetpolynucleotide, and where appropriate, the FLAP probe. The term “FLAPendonuclease” refers to nucleases that recognize FLAP structures and areknown to cleave off the unpaired 5-prime region of the 5-primepolynucleotide probe (e.g., target probe). FLAP endonucleases are alsoknown in some embodiments as FEN-1 (Five' ExoNuclease or FlapEndoNuclease) and “structure specific 5′-exonucleases.” While not beingbound by any theory of action, the FEN-1 nucleases appear to participatein DNA damaged fragment excision, recombinational mismatch correction,and processing of Okazaki fragments during lagging strand DNA synthesis.The endonuclease recognizes and cleaves a double stranded branchednucleic acid structure containing a single-stranded 5-prime flap at thejunction where the two strands of duplex DNA adjoin the single-strandedarm. The FEN-1 nucleases, however, do not appear to act efficiently onbubble substrates, 3-prime single-stranded flaps, heterologous loops,and Holliday junctions. FEN-1 endonucleases obtained from naturalsources typically have an associated 5-prime to 3-prime exonucleaseactivity, which can remove RNA primers during lagging strand synthesisand damaged DNA fragments in various DNA repair pathways. Based onprotein sequence comparison and biochemical assays, two major conservedmotifs, the N (N-terminal) and I (intermediate) motifs, correlate withnuclease activities of FEN-1 type FLAP endonucleases. The FLAPendonuclease activity is not affected by the flap sequence and isgenerally independent of the 5-prime flap length, cleaving a 5-primeflap as small as one nucleotide. FLAP endonucleases are described for,among others, human (P39748; Harrington and Lieber, supra); chimpanzee(XM_508480.1 GI:55636162); dog (XM_533271.2 GI:73983482); mouse(NM_007999.3 GI:47132513; Karanjawala et al., 2000, Microb Comp Genomics5(3):173-7; Emoto et al., 2005, Gene 357 (1):47-54); rat (NM_053430.1GI:16758169; Kim et al., 2000, Biochim Biophys Acta 1496(2-3):333-340);Xenopus laevis (Kim et al., 1998, J Biol Chem 273(15):8842-8; Bibikovaet al., 1998, J Biol Chem 273(51):34222-9.); zebra fish (AY391423.1GI:37362213); Drosophila melanogaster (NP_523765.1 GI:17647423; Ishikawaet al., 2004, Nucleic Acids Res 32 (21):6251-6259); Caenorhabditiselegans (NP_491168.1 GI:17510005) Saccharomyces cerevisiae (Harringtoneand Lieber, supra); Schizosaccharomyces pombe (Alloeva and Doetsch,1998, Nucleic Acids Res 26(16):3645-50); archae (Shen et al.., 1998,Trends Biochem Sci 23(5):171-3; Hwang et al., 1998, Nat Struct Biol5(8):707-13; Hosfield et al.., 1998, J Biol Chem 273(42):27154-27161;Collins et al.., 2004, Acta Crystallogr D Biol Crystallogr60(9):1674-8); Matsui et al., 1999, J Biol Chem 274(26):18297-309; andKaiser et al., 1999, J Biol Chem 274(30):21387-21394), Oryza sativa(Kimura et al., 2003, Gene 314:63-71); phage T5 (Patel et al., 2002, JMol Biol 320(5):1025-35); Arabidopsis thaliana (NP_850877.2GI:42570539); and cauliflower (Kimura et al., 1997, Nucleic Acids Res25(24):4970-6). All publications incorporated herein by reference.

In other embodiments, the FLAP endonucleases are cleavase agents basedon various modified polymerases that recognize and cleave the FLAPstructures. Generally, these modified polymerases have a 5-primenuclease activity but reduced or absent polymerase synthetic activity.FLAP endonucleases of this type have been described for Thermosaquaticus, Thermos flavus, Thermos thermophilus, and Cleavase™ (see,e.g., U.S. Pat. No. 5,541,321 and U.S. Pat. No. 5,614,402).

An illustrative example of a FLAP substrate assay is given in FIG. 4A.The coded molecule in the FLAP assay is similar to the coded moleculedescribed for FIG. 1A, except that the orientation is reversed withrespect to the 5-prime and 3-prime termini. The FLAP structure comprisesa target polynucleotide 403, which has adjacent first and secondregions. The target probe has a 3-prime region 400 that is complimentaryto the first region of the target polynucleotide, and has an unpaired5-prime region 401, which in the illustrated embodiment, comprises asignal generating segment. A FLAP probe 402 is hybridized to the secondregion of the target polynucleotide via a 5-prime region such that the3-prime region of the target probe and the 5-prime region of the FLAPprobe are adjacent to one another when hybridized to the targetpolynucleotide. Upon treatment with a FLAP endonuclease, the targetprobe is cleaved off, thereby resulting in separation of the 5-primeregion of the target probe from the coded molecule. FIG. 4B symbolicallyillustrates current blockade signal pattern of a FLAPendonuclease-modified coded molecule and the signal pattern of anunmodified coded molecule. Loss of the 5-prime region of the targetprobe alters the signal pattern of the FLAP endonuclease modified codedmolecule as compared to the signal pattern of an unmodified codedmolecule.

As discussed herein, detection of a plurality of different targetpolynucleotide can be based on used of a plurality of different codedmolecules in which each coded molecule has a detectable signal patterndistinguishable from the other coded molecules. Upon interrogating thecoded molecule by translocation through a nanopore, the detected signalpattern is decoded and associated to a specific coded molecule and itstarget probe. Typically, the FLAP endonuclease cleavage site is afterthe first or second nucleotide in the hybridized duplex formed by the3-prime polynucleotide probe in the FLAP structure (see, e.g., Kaiser etal., 1999, J Biol Chem 274(23):21387-21394). The presence of a gapbetween the hybridized 5-prime polynucleotide probe and the 3-primepolynucleotide probe (i.e., the two hybridized probes are not abutted toeach other), for example because of a nucleotide mismatch in 3-primeregion of the 5-prime polynucleotide probe, results in inefficientcleavage by the FLAP endonuclease. As such, in some embodiments, thetarget probe is used to interrogate a site of nucleotide sequencevariation on the target polynucleotide.

In other embodiments, the FLAP probe, rather than the target probe, isused to interrogate a site of nucleotide polymorphism on the targetpolynucleotide. In these embodiments, the 3-prime nucleotide of the3-prime region of the FLAP probe interrogates a position of sequencevariation in a target polynucleotide. As above, the presence of amismatch generates a gap between the hybridized FLAP probe and the3-prime region of target probe such that the substrate is notefficiently recognized and cleaved by the FLAP endonuclease.

In various embodiments, the 5-prime region of the target probe cleavedby the FLAP endonuclease can have a capture tag, such as a capturesequence or biotin ligand, which allows the unmodified coded molecule tobe separated from the modified coded molecule because of the removal ofthe capture tag in a FLAP endonuclease-modified coded molecule.

5.3.6 Modification of Coded Molecules by a Sequence Specific or MismatchSpecific Nuclease

In some embodiments, hybridization of the target probe to the targetpolynucleotide forms an endonuclease recognition site that is recognizedby an endonuclease, which cleaves the target probe, thereby modifyingthe coded molecule. As such, in these embodiments, the modifying agentis an endonuclease that specifically recognizes the endonucleaserecognition site. The endonuclease recognition site can be a specificnucleotide sequence and/or a polynucleotide structure. A sequencespecific endonuclease recognizes a sequence formed in the hybrid. Forexample, restriction endonucleases, such as Type II restrictionendonucleases, cleave double stranded DNAs on one or both strands of theduplex DNA by recognizing a specific nucleotide sequence, which can beasymmetric or palindromic. On the other hand, structure specificendonucleases recognize a conformation, such as a mismatched nucleotidepair in a duplex, although there can be some effect on endonucleaseactivity by the type of nucleotide mismatch or the sequences surroundingthe mismatch.

In the embodiments above, the method generally comprises hybridizing atarget probe to a target polynucleotide to generate a recognition sitefor an endonuclease and a corresponding endonuclease cleavage site, andtreating the hybridized polynucleotides with an endonuclease thatrecognizes the recognition site and cleaves the hybridized target probe.In some embodiments, the recognition site formed is a sequence specificendonuclease recognition site and the modifying agent is a sequencespecific endonuclease that recognizes the recognition site and cleavesthe target probe. Removal of a portion of the target probe from thecoded molecule can alter the signal pattern, which can be used todiscern the presence or absence of a target polynucleotide.

The choice of the sequence-specific endonuclease is dependent on therecognition site that is generated upon hybridization of the targetprobe to the target polynucleotide. Recognition sites for various typesof restriction endonucleases useful for the purposes herein, include,among others, Type I, Type II, and Type III restriction endonucleases(Pingoud and Jeltsch, 2001, Nucleic Acids Res. 29(18) 3705-3727). Insome embodiments, the target polynucleotide detected comprises a site ofrestriction site polymorphism, which refers to an endonucleaserecognition site that is present in one target polynucleotide but islacking in a second target polynucleotide. Typically, restriction sitepolymorphisms arc present on target polynucleotides obtained from thesame segment of the chromosome in different members of the same species.This sequence variation forms the basis for restriction fragment lengthpolymorphism (RFLP) used for genotyping analysis. Because of itsspecificity, restriction site polymorphisms based on Type II restrictionenzyme recognition sequences are frequently used for genotypinganalysis, and can be adapted to the methods herein.

In other embodiments, the endonuclease recognition site formed byhybridization of the target probe to the target polynucleotide is anucleotide mismatch, which is recognized by a mismatch-specificendonuclease. A “nucleotide mismatch” refers to the pairing ofnon-complementary bases when a target polynucleotide hybridizes toanother polynucleotide. A single nucleotide mismatch is present when atarget polynucleotide and another polynucleotide are complementaryexcept for a single non-complementary base-pair. Mismatches can resultfrom pairing of a purine with another purine (e.g., A paired with A, Gpaired with G, A paired with G), a pyrimidine with another pyrimidine(e.g., C paired with C, C paired with T, and T paired with T), and apyrimidine with a purine (e.g., C paired with A, and T paired with G).In the embodiments herein, the presence of target polynucleotides isdetected by hybridizing a target probe to a target polynucleotide andtreating with an endonuclease that recognizes any mismatches, whichleads to cleavage of the target probe if a mismatch is present. Perfectcomplementarity between the target probe and the target polynucleotidedoes not lead to modification of the target probe.

Various mismatch recognizing endonucleases and mismatch repair systemshave been described (see, e.g., U.S. Pat. No. 5,869,245) and can be usedfor the methods herein. The Mut system is mismatch detection systemfound in some prokaryotes, which in E. coli. comprises proteins MutH,MutS, and MutL (Smith et al., 1996, Proc Natl Acad Sci USA93:4374-4379). The MutS protein recognizes a mismatch and associateswith MutL to form a complex that activates the latent endonucleaseactivity of MutH. MutH cleaves on one side of the mismatch at ahemi-methylated (GATC) sequence and must be activated by MutS and MutLto cleave the DNA. Similar systems have been identified in yeast andmammalian cells and can be adapted for the methods herein.

Another useful mismatch endonuclease is E. coli. vsr endonuclease, whichrecognizes G:T base-pair mismatches in double-stranded DNA within thesequence (CC[A/T]GG) and initiates a repair pathway by hydrolyzing thephosphate group 5-prime to the incorrectly paired T (Turner andConnelly, 2000, J Mol Biol. 304(5):765-78). In other embodiments, themismatch endonuclease is Uvelp, which recognizes all potential DNA basemispair combinations. This endonuclease, found in Schizosaccharomycespombe, recognizes and cleaves DNA 5-prime to the mispaired base in astrand-specific manner. (Kauer et al., 1999, Mol Cell Biol19(7):4703-4710). Uvelp exhibits strong cleavage at the *C/C, *C/A, and*G/G sites; moderate cleavage at *G/A, *A/G, and *T/G sites, and weakcleavage at *G/T, *A/A, *A/C, *C/T, *T/T, and *T/C sites (* indicatesthe cleaved strand in the mismatch). Thus, Uvelp is useful for detectingmost types of mismatches formed between the target probe and the targetpolynucleotide.

Other types of mismatch endonucleases with broad specificity can beobtained from plants, such as celery. Plant mismatch endonucleases ofthis type are characterized by an activity with a neutral pH optima andare capable of detecting destabilized regions of DNA helices, such as ata site of a mismatch (Olekowski et al., Nucleic Acids Res26(20):4597-4602). An exemplary embodiment of a plant mismatchendonuclease is CEL-1 from celery, a mismatch endonuclease thatrecognizes base substitution mismatched substrates and cuts on one ofthe two DNA strands for each mismatch duplex. The activity of CEL 1 canbe enhanced by the presence of Taq DNA polymerase.

In various embodiments, the length of the target probe cleaved from thecoded molecule can be such that the modification alters the signalpattern of the modified coded molecule. The length of the targetpolynucleotide can be varied, with or without adjusting the length ofthe region complementary to the target polynucleotide. Thus in someembodiments, the target probe can have a non-complementary region, suchas a signal generating segment, that is cleaved off by action of theendonuclease.

An illustrative embodiment of a sequence-specific or structure specificendonuclease assay is provide in FIG. 5A. The target probe 501hybridizes to a target polynucleotide 504 to generate an endonucleaserecognition site 502. In the illustrated embodiment, the endonucleasecleavage site is included within the endonuclease recognition site. Upontreatment with an endonuclease, the target probe is cleaved at theendonuclease cleavage site, thus separating a portion of the targetprobe from the coded molecule and modifying the signal pattern of thecoded molecule. In the illustrated embodiment, the target probe furthercomprises a signal generating segment 503 to distinguish theendonuclease modified coded molecule from the unmodified coded molecule.FIG. 5B symbolically illustrates a current blockade signal pattern foran endonuclease-modified coded molecule and the signal pattern of anunmodified coded molecule.

5.3.7 Modification of Coded Molecules by Double Stranded SpecificExonucleases

In some embodiments, the modification is target polynucleotide directeddegradation of the target probe. In these embodiments, hybridization ofthe target probe to a target polynucleotide forms a double strandedsegment, which is then treated with a suitable exonuclease havingspecificity for a double stranded polynucleotide. “Exonuclease” refersto a nuclease that degrades a polynucleotide starting from a terminus ofthe polynucleotide and progressing inward. Double stranded exonucleasesdegrade the target probe hybridized to the target polynucleotide, aswell as the target polynucleotide if susceptible to degradation, therebymodifying the coded molecule. If a sufficient amount of the target probeis degraded, a signal pattern that differs from a non-degraded codedmolecule can be generated. Integrity of the coded molecule, other thantarget probe portion, can be protected by use of nucleobase polymersresistant to nuclease activity (e.g., polynucleotide analogs having PNAbackbones).

In some embodiments, the double-stranded exonuclease is a 5-prime to3-prime double stranded dependent exonuclease and the target probecomprises a 5-prime terminal region that hybridizes to the targetpolynucleotide. Suitable 5-prime to 3-prime specific double strandedexonucleases include, among others, λ exonuclease (Higuchi and Ochman,1989, Nucl. Acids Res 17:5865, T4 exonuclease B, T7 gene 6 exonuclease(Engler and Richardson, 1983, J Biol. Chem 258, 11197-11205), mammalianDNase IV (also known as FEN-1), exonuclease VI (pol 1 small fragment),and exonuclease VII. Where the 5-prime region of the target probehybridizes to the target polynucleotide but presents a single-strandedoverhang, nucleases acting on the single stranded overhang can be usedto make the hybridized complex susceptible to the 5-prime to 3-primedouble stranded exonucleases.

In other embodiments, the double-stranded exonuclease is a 3-prime to5-prime double stranded exonuclease and the target probe comprises a3-prime terminal region that hybridizes to a target polynucleotide toform a double stranded polynucleotide. Exemplary exonucleases of thistype include, among others, exonuclease III (also referred to as APendonuclease VI) and 3-prime to 5-prime nuclease activity of DNApolymerases. As above, where the 3-prime terminal region of the targetprobe hybridizes to the target polynucleotide but forms asingle-stranded overhanging segment, nucleases acting on the singlestranded overhang can be used to make the hybridized complex susceptibleto the 3-prime to 5-prime double stranded exonuclease.

In some embodiments, the length of the target probe removed by treatmentwith exonuclease can be varied by adjusting the length of thecomplementary region between the target probe and the targetpolynucleotide. In various embodiments, the amount of target probedegraded can be about 10 nucleotides, about 20 nucleotides, about 50nucleotides, about 100 nucleotides, about 200 nucleotides, about 500nucleotides, up to about 1000 nucleotides or more. It is well within theskill of those in the art to determine the amount that can be removedfrom the target probe to alter the signal pattern of the coded molecule.

In some embodiments, where the target polynucleotide is an amplifiedproduct, such as PCR product of a nucleic acid of interest, the targetpolynucleotide can be rendered nuclease resistant, thus limiting thenuclease mediated degradation to the target probe. In still otherembodiments, the target probe can be a chimeric polynucleotide in whichone region is nuclease resistant while another region is susceptible tothe nuclease. Such chimeric target probes can provide a specificendpoint for degradation, thereby providing a well defined signalpattern for the modified coded molecule.

5.3.8 Multiplexing Formats

The methods of detecting a target polynucleotide described in theforegoing sections are adaptable for use in multiplex formats to detecta plurality of different target polynucleotides in a single reaction. Insome embodiments, coded molecules with differing signal patterns areused for multiplexed detection, where each coded molecule with adistinguishable signal pattern has a target probe that is complementaryto a sequence of a specific target polynucleotide. The plurality ofdifferent coded molecules are contacted with a sample, and treated witha modifying agent that modifies the coded molecule in a targetpolynucleotide dependent manner. Coded molecules are then translocatedthrough a nanopore to detect a signal pattern, which is associated to aspecific coded molecule and thus the target polynucleotide detected bythat coded molecule. As noted herein, in some embodiments, the modifiedcoded molecule can be isolated away from unmodified coded molecules viaa capture tag on the coded molecule prior to translocation through ananopore, either to increase the sensitivity of the detection or toeliminate the need to determine a change in the signal pattern of thecoded molecule.

In various embodiments, the multiplexing method comprises: contacting atleast a first coded molecule and a second coded molecule with aplurality of target polynucleotides, wherein (i) the first codedmolecule comprises a first one or more block polymer regions and a firsttarget probe capable of hybridizing to a first target polynucleotide,wherein the first coded molecule has a detectable first signal pattern,and (ii) the second coded molecule comprises a second one or more blockpolymer regions and a second target probe capable of hybridizing to asecond target polynucleotide, wherein the second coded molecule has adetectable second signal pattern distinguishable from the first signalpattern. Following treatment with a modifying agent that modifies thefirst and second target probes when target polynucleotides arehybridized to the target probes, the coded molecules are translocatedthrough a nanopore to detect the signal pattern of each coded molecule.The detected signal pattern is associated to the first or second codedmolecule to determine the presence or absence of the targetpolynucleotides.

Similarly, in some embodiments for detecting a plurality of targetpolynucleotides, a population of coded molecules is contacted with aplurality of target polynucleotides, wherein the population of codedmolecules comprises a plurality of subpopulations and each codedmolecule of each subpopulation comprises: (i) a plurality of blockpolymer regions and a target probe capable of hybridizing to a targetpolynucleotide, wherein the target probe of each subpopulationhybridizes to a different target polynucleotide; and (ii) a detectablesignal pattern distinguishable amongst the plurality of subpopulations.The target probe is modified with a modifying agent, wherein themodification is dependent on the presence of a target polynucleotidehybridized to the target probe. The coded molecule is then translocatedthrough a nanopore to detect the signal pattern of each of the codedmolecules. As above, the detected signal pattern is associated to aspecific subpopulation of coded molecules to determine the presence orabsence of a specific target polynucleotide.

In still other embodiments, multiplexed detection of a plurality ofdifferent target polynucleotides can be based on modifications thatchange the signal pattern of the coded molecule, where the change in thesignal pattern is different for each different target nucleotidedetected. In some embodiments, the multiplexed detection uses aplurality of different ligation probes as discussed above, where eachligation probe hybridizes to a different target polynucleotide and eachligation probe has a distinguishable signal pattern. In theseembodiments, the distinguishable signal pattern of the ligation probes,such as that formed by a signal generating segment, can be used todifferentiate the detection of one target polynucleotide from anothertarget polynucleotide.

In other embodiments, the multiplexed detection uses elongation of the3-prime terminal region of a target probe in a rolling circlereplication assay. In these embodiments, a plurality of differentcircular target polynucleotides (i.e., ligated open circle probes (OPC))can be used, where each circular target polynucleotide has adistinguishable signal pattern and each hybridizes with a differentnucleic acid of interest in forming the ligated OCP. Detecting aspecific signal pattern associated with the replicated circular targetpolynucleotide indicates the presence or absence of a specific circulartarget polynucleotide and thus the presence or absence of a specificnucleic acid of interest.

In still other embodiments, various coded molecules with differingsignal patterns can be used in combination with modifications thatprovide different signal generating segments, such as with ligationprobes or rolling circle templates, to generate a large number ofdistinguishable signal pattern combinations for detecting a plurality ofdifferent target polynucleotides. For instance, if five different SNPsare known to occur for a gene sequence, each occurring at differentpositions on the gene, five coded molecules with differing signalpatterns can be made, one for each SNP site, and combined with fivepairs of ligation probes, each pair selective for a normal and variantsequence at one SNP site. Each ligation probe pair can use the same pairof distinguishable signal generating segments, e.g., a first signalgenerating segment for the ligation probe detecting the normal sequenceand a second signal generating segment for the ligation probe detectingthe variant sequence. The same pair of signal generating segments can beused for each ligation probe pair since the signal pattern from thecombination of the coded molecule and the ligation probe allowsdifferentiation all ten different possible products. As will be apparentfrom the foregoing, a large number of different combinations can begenerated.

5.3.9 Preparation of Coded Molecules, Ligation Probes, and FLAP Probes

The coded molecules, ligations probes, FLAP probes, and other polymerscan be made by standard methodologies known in the art. The codedmolecule can be synthesized in whole or in parts, where the parts aresubsequently joined together. Polynucleotides can be synthesized usingstandard chemistries, such as phosphoramiditc chemistries (see, e.g.,Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, 2003;U.S. Pat. No. 4,973,679; Beaucage, 1992, Tetrahedron 48:2223-2311; U.S.Pat. No. 4,415,732; U.S. Pat. No. 4,458,066; U.S. Pat. No. 5,047,524 andU.S. Pat. No. 5,262,530; all of which are incorporated herein byreference). Chimeric polynucleotides, for example chimeras of PNA andDNA, are described in various references, such as U.S. Pat. No.6,297,016. Synthesis can be carried out using automated synthesizersavailable commercially, for example the Model 392, 394, 3948 and/or 3900DNA/RNA synthesizers available from Applied Biosystems, Foster City,Calif.

Methods for synthesizing polynucleotide analogs and mimics will alsofollow standard methodologies. For example, PNAs are described in U.S.Pat. Nos. 5,539,082; 5,527,675; 5,623,049; 5,714,331; 5,718,262;5,736,336; 5,773,571; 5,766,855; 5,786,461; 5,837,459; 5,891,625;5,972,610; 5,986,053; 6,107,470; 6,201,103; 6,350,853; 6,357,163;6,395,474; 6,414,112; 6,441,130; and 6,451,968; all of which are hereinincorporated by reference. General description for PNA synthesismethodologies is given in Nielsen et al., 1999, Peptide Nucleic Acids;Protocols and Applications, Horizon Scientific Press, Norfolk England.

Where the coded molecule is a composite of non-nucleobase and nucleobasepolymers, the coded molecule can be synthesized in segments and thenassembled together or, alternatively, formed by sequential synthesis ofthe non-nucleobase polymer region and the nucleobase polymer region. Forexample, phosphoramidite polyethylene glycols along with phosphoramiditenucleotides for synthesis of nucleic acid-PEG composite polymers(Sanchez-Quesada et al., supra) can be used as precursors forsynthesizing a composite polymer of polynucleotides and PEG.

Recombinant techniques may also be used to synthesize the codedmolecule, or part thereof (see, e.g., Sambrook et al., supra; Ausuble etal., supra). For instance, single-stranded polynucleotides are readilymade using single-stranded phage systems by cloning block polymerregions and replicating single-stranded copies of the clonedpolynucleotides. Alternatively, polynucleotide sequences forming thecoded molecule can be inserted into an appropriate expression vehicle,i.e., a vector which contains the necessary elements for transcription(e.g., T4 or T7 RNA polymerase systems), or in the case of an RNA viralvector, the necessary elements for replication of the RNA. Theexpression vehicle is then transfected into a suitable host cell whichcan express the nucleic acid, or used in in vitro transcription systemsfor synthesis of the desired polynucleotide. Depending on the expressionsystem used, the expressed polynucleotide is then isolated by procedureswell-established in the art. Methods for recombinant production ofpolynucleotides are well established and can be found in standardreferences such as Sambrook et al., 2001, Molecular Cloning A LaboratoryManual, 3^(rd) Ed., Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y., updates to 2005, thedisclosures of which is incorporated herein in its entirety.

5.4 Nanopore Devices

In the present disclosure, detecting the coded molecules is carried outby translocating the coded molecule through a nanopore or nanochannel.As used herein, a “pore” or “channel” refers to an orifice, gap,conduit, or groove of sufficient dimension that allows passage oranalysis of a single coded molecule. In some embodiments, the nanoporeor channel is dimensioned for translocation of not more that one codedmolecule at a time. Thus, the dimensions of the nanopore in someembodiments will typically depend on the dimensions of the codedmolecule to be examined. A code molecule with a double-stranded regioncan require a nanopore dimension greater than those sufficient fortranslocation of a coded molecule which is entirely single-stranded. Inaddition, presence of detectable tags or capture tags can require largerpores or channels than coded molecules lacking such tags. Typically, apore of about 1 nm diameter can permit passage of a single strandedpolymer, while pore dimensions of 2 nm diameter or larger will permitpassage of a double-stranded nucleic acid molecule. In some embodiments,the nanopore or nanochannel is selective for a single stranded codedmolecule (e.g., from about 1 nm to less than 2 nm diameter) while inother embodiments, the nanopore or nanochannel is of a sufficientdiameter to permit passage of double stranded polynucleotides (e.g., 2nm or larger). As noted above, it is to be understood that the pore orchannel dimensions may be larger where the detection method sufficientlydiscriminates single coded molecules passing through the pore and/orwhere the coded molecule has dimensions larger than a double-strandedpolynucleotide. For example, for detection based on electron tunneling,the detection region is spatially within a few nanometers of the codedmolecule such that the pore or channel is not much larger than the codedmolecule itself However, some detection techniques do not requirenanometer proximity of the coded molecule to the detection region andmay be capable of detecting a single coded molecule even when the pore,conduit, channel or groove is significantly larger than that requiredfor electron tunneling detection (see, e.g., U.S. Pat. No. 6,413,792 andU.S. published application No. 2003/0211502, incorporated herein byreference).

Various types of nanopore may be used for analyzing the coded molecules.These include, among others, biological nanopores that employ abiological pore or channel embedded in a membrane. Another type ofnanodevice is a solid state nanopore in which the channel or pore ismade whole or in part from a fabricated or sculpted solid statecomponent, such as silicon.

5.4.1 Biological Pores

For detecting the coded molecules, any biological pore with channeldimensions that permit translocation of the coded molecules can be used.Two broad categories of biological channels are suitable for the methodsdisclosed herein. Non-voltage gated channel allow passage of moleculesthrough the pore without requiring a change in the membrane potential toactivate or open the channel. On the other hand, voltage gated channelsrequire a particular range of membrane potential to activate channelopening. Most studies with biological nanopores have used a-hemolysin, amushroom-shaped homo-oligomeric heptameric channel of about 10 nm inlength found in Staphylococcus aureus. Each subunit contributes two betastrands to form a 14 strand anti-parallel beta barrel. The pore formedby the beta barrel structure has an entrance with a diameter ofapproximately 2.6 nm that contains a ring of lysine residues and opensinto an internal cavity with a diameter of about 3.6 nm. The stem of thehemolysin pore, which penetrates the lipid bilayer, has an averageinside diameter of about 2.0 nm with a 1.5 nm constriction between thevestibule and the stem. The dimensions of the stem are sufficient forpassage of single-stranded nucleic acids but not double-stranded nucleicacids. Thus, α-hemolysin pores may be used as a nanopore selective forsingled-stranded polynucleotides and other polymers of similardimensions.

In other embodiments, the biological nanopore is of a sufficientdimension for passage of polymers larger than a single-stranded nucleicacid. An exemplary pore is mitochondrial porin protein, a voltagedependent anion channel (VDAC) localized in the mitochondrial outermembrane. Porin protein is available in purified form and, whenreconstituted into artificial lipid bilayers, generates functionalchannels capable of permitting passage of double-stranded nucleic acids(Szabo et al., 1998, FASEB J. 12:495-502). Structural studies suggestthat porin also has a beta-barrel type structure with 13 or 16 strands(Rauch et al., 1994, Biochem Biophys Res Comm 200:908-915). Porindisplays a larger conductance compared conductance of pores formed byα-hemolysin, maltoporin (LamB), and gramicidin. The larger conductanceproperties of porin support studies showing that the porin channel issufficiently dimensioned for passage of double-stranded nucleic acids.Pore diameter of the porin molecule is estimated at 4 nm. The diameterof an uncoiled double-stranded nucleic acid is estimated to be about 2nm.

Another biological channel that may be suitable for scanning doublestranded polynucleotides are channels found in B subtilis (Szabo et al.,1997, J. Biol. Chem. 272:25275-25282). Plasma membrane vesicles madefrom B subtilis and incorporated into artificial membranes allow passageof double-stranded DNA across the membrane. Conductance of the channelsfaulted by B subtilis membrane preparations is similar to those ofmitochondrial porin. Although there is incomplete characterization(e.g., purified form) of these channels, it is not necessary to havepurified forms for the purposes herein. Diluting plasma membranepreparations, either by solubilizing in appropriate detergents orincorporating into artificial lipid membranes of sufficient surfacearea, can isolate single channels in a detection apparatus. Limiting theduration of contact of the membrane preparations (or proteinpreparations) with the artificial membranes by appropriately timedwashing provides another method for incorporating single channels intothe artificial lipid bilayers. Conductance properties may be used tocharacterize the channels incorporated into the bilayer.

In some embodiments, the biological pore may be modified to incorporatea sensing label for sensing the detectable property of the codedmolecule, including the sensing of detectable tags incorporated into thecoded molecule. Various sensing labels may be used to modify the channelof the biological pore but without significantly affecting the channeldimensions. For example, α-hemolysin has been modified at the poreregion by attachment of a short single-stranded nucleotide (via alinker) to a cysteine residue on the hemolysin channel subunit. Poreswith modifications to only one of the pore subunits can alter thetranslocation of single-stranded nucleic acids through the hemolysinpore. Single-stranded molecules that hybridize to the attachednucleobase oligomer display current blockades longer in duration thansingle-stranded nucleic acids that are not complementary to the attachednucleobase oligomer (Howorka et al., 2001, Nature Biotechnol.18:1091-5).

For generating the biological nanopores, proteins capable of forming thechannels can be isolated from natural sources or made by recombinantmethods (Szabo et al., supra; Sambrook et al., supra; Ausubel et al.,supra). In other embodiments, isolated plasma membrane preparations canbe used as the source of the biological pores. Proteins can bereconstituted into artificial membranes and functional channels detectedusing standard electrophysiological techniques used to measure singlechannel activity. Methods and apparatus for incorporating biologicalpores into artificial membranes are described in U.S. Pat. No.6,267,872, incorporated herein by reference.

5.4.2 Solid State Pores

In other embodiments, analysis of the coded molecules is carried out bytranslocating the coded molecule through a nanopore or nanochannelfabricated from non-biological materials. Nanopores or channels can bemade from a variety of solid state materials using a number of differenttechniques, including, among others, chemical deposition,electrochemical deposition, electroplating, electron beam sculpting, ionbeam sculpting, nanolithography, chemical etching, laser ablation, andother methods well known in the art (see, e.g., Li et al., 2001, Nature412:166-169; and WO 2004/085609). Solid state materials include, by wayof example and not limitation, any known semiconductor materials,insulating materials, and metals. Thus, the nanopores may comprisewithout limitation silicon, silicon, silicon nitride, germanium, galliumarsenide, metals (e.g., gold, silver, platinum), metal oxides, and metalcolloids.

To make a pore of nanometer dimensions, various feedback procedures canbe employed in the fabrication process. In embodiments where ions passthrough a hole, detecting ion flow through the solid state materialprovides a way of measuring pore size generated during fabrication (see,e.g., U.S. Published Application No. 2005/0126905). In otherembodiments, where the electrodes define the size of the pore, electrontunneling current between the electrodes gives information on the gapbetween the electrodes. Increases in tunneling current indicate adecrease in the gap space between the electrodes. Other feedbacktechniques will be apparent to the skilled artisan.

In some embodiments, the nanopore is fabricated using ion beamsculpting, as described in Li et al., 2003, Nature Materials 2:611-615.In the described process, a layer of low stress silicon nitride film isdeposited onto a silicon substrate via low pressure chemical vapordeposition. A combination of photolithography and chemical etching canbe used to remove the silicon substrate to leave behind the siliconnitride layer. To form the pore, a focused ion beam (e.g., argon ionbeam of energy 0.5 to 5.0 KeV and diameter 0.1 to 0.5 mm) is used togenerate a hole in the silicon nitride membrane. By suitable adjustmentof the ion beam parameters (e.g., total time the silicon nitride isexposed to the ion beam and the exposure duty cycle) and sampletemperature, material can be either removed to enlarge the hole ormaterial added to decrease the hole size. Ion beam bombardment at roomtemperature and low duty cycle results in migration of material into thehole while bombardment at 5° C. and longer duty cycles results inenlargement of the hole. Measuring the amount of ions transmittedthrough the pore provides a feedback mechanism for precisely controllingthe final pore size (Li et al., supra). To form a nanopore of usefuldimensions, a hole larger than the final desired pore dimensions can bemade using sculpting parameters that result in loss of the siliconnitride. Subsequently, the size of the pore is adjusted to a dimensionsuitable for translocation of non-single-stranded polymers usingsculpting parameters that result in movement of material into theinitially formed hole.

In other embodiments, the nanopores may be made by a combination ofelectron beam lithography and high energy electron beam sculpting (see,e.g., Storm et al., 2003, Nature Materials 2:537-540). Asilicon-on-insulator, fabricated according to known methods, is used toform a silicon membrane, which is then oxidized to form a silicon oxidelayer. Using a combination of electron-beam lithography and anisotropicetching, the silicon oxide is removed to expose the silicon layer. Holesare made in the silicon by KOH wet etching and the silicon oxidized toform a silicon oxide layer of about 40 nm. Exposure of the silicondioxide to a high energy electron beam (e.g., from a transmissionelectron microscope) deforms the silicon dioxide layer surrounding thehole. Whether the initial holes are enlarged or decreased depends on theinitial size. Holes 50 nm or smaller appear to decrease in size whileholes of about 80 nm or larger increase in size. A similar approach forgenerating a suitable nanopore by ion beam sputtering technique isdescribed in Heng et al., 2004, Biophy J 87:2905-2911. The nanopores areformed using lithography with a focused high energy electron beam onmetal oxide semiconductor (CMOS) combined with general techniques forproducing ultrathin films.

In other embodiments, the nanopore is constructed as provided in U.S.Pat. No. 6,627,067; 6,464,842; 6,783,643; and U.S. Publication No.2005/0006224 by sculpting of silicon nitride. Initially, a layer ofsilicon nitride is deposited on both sides of a silicon layer bychemical vapor deposition. Following addition of a photoresist in amanner that leaves a portion of the silicon nitride layer exposed, theexposed silicon nitride layer on one side is removed by conventional ionetching techniques to leave behind a silicon coated with silicon nitrideon the other side. The silicon can be removed by any number of etchingtechniques, such as by anisotropic KOH etching, thus leaving behind amembrane of silicon nitride. The thickness of the silicon nitridemembrane is controlled by adjusting the thickness deposited onto thesilicon. By use of electron beam lithography or photolithography, acavity is produced on one side of the silicon nitride layer followed bythinning of the membrane on the other side of the cavity. Suitablethinning processes include, among others, ion beam sputtering, ion beamassisted etching, electron beam etching, and plasma reactive etching.Numerous variations on this fabrication process, for example, use ofsilicon nitride layer sandwiched between two silicon layers, can be usedto generate different nanopores. As noted above, a feedback mechanismbased on measuring the rate and/or intensity of ions passing through thepore provides a method of controlling the pore size during thefabrication process.

In still other embodiments, the nanopore can be constructed as a gold orsilver nanotube. These nanopores are formed using a template of porousmaterial, such as polycarbonate filters prepared using a track etchmethod, and depositing gold or other suitable metal on the surface ofthe porous material. Track etched polycarbonate membranes are typicallyformed by exposing a solid membrane material to high energy nuclearparticles, which creates tracks in the membrane material. Chemicaletching is then employed to convert the etched tracks to pores. Theformed pores have a diameter of about 10 nm and larger. Adjusting theintensity of the nuclear particles controls the density of pores formedin the membrane. Nanotubes are formed on the etched membrane bydepositing a metal, typically gold or silver, into the track etchedpores via an electroless plating method (Menon et al., 1995, Anal Chem67:1920-1928). This metal deposition method uses a catalyst deposited onthe surface of the pore material, which is then immersed into a solutioncontaining Au(I) and a reducing agent. The reduction of Au(I) tometallic Au occurs on surfaces containing the catalyst. Amount of golddeposited is dependent on the incubation time such that increasing theincubation time decreases the inside diameter of the pores in the filtermaterial. Thus, the pore size may be controlled by adjusting the amountof metal deposited on the pore. The resulting pore dimension is measuredusing various techniques, for instance, gas transport properties usingsimple diffusion or by measuring ion flow through the pores using patchclamp type systems. The support material is either left intact, orremoved to leave gold nanotubes. Electroless plating technique iscapable of forming pore sizes from less than about 1 nm to about 5 nm indiameter, or larger as required. Gold nanotubes having pore diameter ofabout 0.6 nm appears to distinguish between Ru(bpy)2+2 and methylviologen, demonstrating selectivity of the gold nanopores (Jirage etal., 1997, Science 278:655-658). Modification of a gold nanotube surfaceis readily accomplished by attaching thiol containing compounds to thegold surface or by derivatizing the gold surface with other functionalgroups. This features permits attachment of pore modifying compounds aswell as sensing labels, as discussed herein. Devices, such as thecis/trans apparatuses used for biological pores described herein, can beused with the gold nanopores to analyze single coded molecules.

Where the mode of detecting the coded molecule involves current flowthrough the coded molecule (e.g., electron tunneling current), the solidstate membrane may be metalized by various techniques. The conductivelayer may be deposited on both sides of the membrane to generateelectrodes suitable for interrogating the coded molecule along thelength of the chain, for example, longitudinal electron tunnelingcurrent. In other embodiments, the conductive layer may be deposited onone surface of the membrane to form electrodes suitable forinterrogating coded molecules across the pore, for example, transversetunneling current. Various methods for depositing conductive materialsare known, including, sputter deposition (i.e., physical vapordeposition), non-electrolytic deposition (e.g., colloidal suspensions),and electrolytic deposition. Other metal deposition techniques arefilament evaporation, metal layer evaporation, electron-beamevaporation, flash evaporation, and induction evaporation, and will beapparent to the skilled artisan.

In some embodiments, the detection electrodes are formed by sputterdeposition, where an ion beam bombards a block of metal and vaporizesmetal atoms, which are then deposited on a wafer material in the form ofa thin film. Depending on the lithography method used, the metal filmsarc then etched by means of reactive ion etching or polished usingchemical-mechanical polishing. Metal films may be deposited on preformednanopores or deposited prior to fabrication of the pore.

In some embodiments, the detection electrodes are fabricated byelectrodeposition (see, e.g., Xiang et al., 2005, Angew. Chem. Int. Ed.44:1265-1268; Li et al., Applied Physics Lett. 77(24):3995-3997; andU.S. Publication Application No. 2003/0141189). This fabrication processis suitable for generating a nanopore and corresponding detectionelectrodes positioned on one face of the solid state film, such as fordetecting transverse electron tunneling. Initially, a conventionallithographic process is used to form a pair of facing electrodes on asilicon dioxide layer, which is supported on a silicon wafer. Anelectrolyte solution covers the electrodes, and metal ions are depositedon one of the electrodes by passing current through the electrode pair.Deposition of metal on the electrodes over time decreases the gapdistance between the electrodes, creating not only detection electrodesbut a nanometer dimensioned gap for translocation of coded molecules.The gap distance between the electrodes may be controlled by a number offeedback processes. In some configurations, the feedback for controllingthe distance between the two electrodes uses the potential differencebetween the two electrodes. As the gap between the electrodes decreases,the potential difference decreases. In other configurations, control ofthe distance between the two electrodes uses the electron tunnelingcurrent across the electrode pair (Li et al, supra). As the distancebetween the electrodes decrease, electron tunneling current increases.Feedback control using electron tunneling is suitable for fabrication ofelectrodes with gap distances of about 1 nm or less, while the feedbackcontrol based on electrode gap potential allows fabrication ofelectrodes having gap distances about 1 to about 10 nm. Rate ofelectrodeposition depends on the electrolyte concentration and thecurrent flowing through the electrodes. Constant current may be used toform layers of metal on the electrodes. In other embodiments, pulses ofcurrent may provide precise control over electrode fabrication sincepulsed currents can be used to deposit a known number of metal atomsonto the electrodes per each pulse cycle.

Where the detection is based on imaging of charge induced field effects,a semiconductor can be fabricated as described in U.S. Pat. No.6,413,792 and U.S. published application No. 2003/0211502. The methodsof fabricating these nanopore devices can use techniques similar tothose employed to fabricate other solid state nanopores. In someembodiments, the field effect detector is made using asilicon-on-insulator that comprises a silicon substrate with a silicondioxide layer and a p-type silicon layer (doped silicon in which themajority of the charge carriers are positively charged holes). A shallown-type silicon (doped silicon in which the majority of the chargecarriers are negatively charged holes) layer is formed in the p-typesilicon layer by ion implantation and addition of an n-type dopant,while another n-type silicon layer that extends through the p-typesilicon layer is formed on another region of the silicon-on-insulator.Removal of the silicon substrate and silicon dioxdc layers by etchingexposes the p-type silicon on the face opposite to the first formedshallow n-type layer. On the newly exposed face of the p-type silicon, asecond shallow n-type silicon layer is formed, which connects to then-type silicon layer that extends through the p-type silicon layer. Forinterrogating the coded molecules, a nanopore that extends through thetwo shallow n-type silicon layers and the p-type silicon layer isgenerated by various techniques, for example by ion etching orlithography (e.g., optical or electron beam). To decrease the nanoporesize, a silicon dioxide layer can be formed by oxidizing the silicon.Metal layers are attached to the first formed n-type silicon layer andthe n-type silicon layer that extends through p-type silicon, therebyforming the source and drain regions. Detection of the coded molecule,and where suitable, the target polynucleotide, is carried out as furtherdescribed below.

For analysis of the coded molecules, the nanopore may be configured invarious formats. In some embodiments, the device comprises a membrane,either biological or solid state, containing the nanopore held betweentwo reservoirs, also referred to as cis and trans chambers (see, e.g.,U.S. Pat. No. 6,627,067). A conduit for electron migration between thetwo chambers allows electrical contact of the two chambers, and avoltage bias between the two chambers drives translocation of the codedmolecule through the nanopore biological nanopores. A variation of thisconfiguration is used in analysis of current flow through biologicalnanopores, as described in U.S. Pat. Nos. 6,015,714 and 6,428,959; andKasianowiscz et al., 1996, Proc Natl Acad Sci USA 93:13770-13773, thedisclosures of which are incorporated herein by reference.

Variations of the device above is disclosed in U.S. applicationpublication no. 2003/0141189. A pair of nanoelectrodes fabricated byelectrodeposition are positioned on a substrate surface. The electrodesface each other and have a gap distance sufficient for passage of asingle nucleic acid. An insulating material protects the nanoelectrodes,exposing only the tips of the nanoelectrodes for the detection of thenucleic acid. The insulating material and nanoelectrodes separate achamber serving as a sample reservoir and a chamber to which the polymeris delivered by translocation. Cathode and anode electrodes provide anelectrophoresis electric field for driving the coded molecule from thesample chamber to the delivery chamber.

The current bias used to drive the coded molecule through the nanoporecan be generated by applying an electric field directed through thenanopore. In some embodiments, the electric field is a constant voltageor constant current bias. In other embodiments, the movement of thecoded molecule is controlled through a pulsed operation of theelectrophoresis electric field parameters (see, e.g., U.S. Pat.Application No. 2003/141189 and U.S. Pat. No. 6,627,067). Pulses ofcurrent may provide a method of precisely translocating one or only afew bases of a coded molecule for a defined time period through the poreand to briefly hold the nucleic within the pore, and thereby providegreater resolution of the electrical properties of the coded molecule.

The nanopore devices may further comprise an electric or electromagneticfield for restricting the orientation of the coded molecule as it passesthrough the nanopore. This holding field can be used to decrease themovement of the coded molecule within the pore. Variations in theposition of the coded molecule in the nanopore can increase thebackground noise of the detected signal. For instance, when currentblockade is measured, movement of the coded molecule within the pore islikely to result in variations of current flow depending on the positionof the coded molecule in the pore. Similarly, where the detectionmeasures electron tunneling current, the current signal is likely to besensitive to the spatial orientation of the coded molecule relative tothe detection electrodes. Movement of the coded molecule, for instancethrough random Brownian motion, would generate variability in the signalmeasured, which may create complexities in assigning a signal pattern toa specific coded molecule. By holding or restricting the orientation ofthe coded molecule as it translocates through the nanopore, variationsin the detected signal can be minimized.

In some embodiments, an electric field that is orthogonal to thedirection of translocation is provided to restrict the movement of thecoded molecule within the nanopore. This is illustrated in U.S.Application Publication No. 2003/0141189 through the use of two parallelconductive plates above and beneath the sample plate. These electrodesgenerate an electric field orthogonal to the direction of translocationof a coded molecule, and thus holding the coded molecule to one of thesample plates. A negatively charged backbone of a DNA, or nucleic acidmodified to have negative charges on one strand, will be oriented ontothe anodic plate, thereby limiting the motion of the coded molecule.Analogous use of an orthogonal electric field to hold a nucleic acid ina limited orientation for detection is described in U.S. Pat. No.6,627,067. Electrodes positioned to generate an electric fieldorthogonal to an extended nucleic acid are used to hold the nucleic acidin a groove, where the nucleic acid is interrogated with a probe (e.g.,electron tunneling probe). Similar to the control of the electric fieldfor moving the coded molecule through the nanopore, the orthogonalelectric field may be controlled in regard to the duration and amplitudeof the holding field. The electric field used for translocation iscoordinated with the electric field used to hold the DNA in a restrictedorientation to precisely control the movement of a nucleic acid throughthe nanopore.

In still other embodiments, controlling the position of the codedmolecule is carried out by the method described in U.S. ApplicationPublication No. 2004/0149580, which employs an electromagnetic fieldcreated in the pore via a series of electrodes positions near or on thenanopore. In these embodiments, one set of electrodes applies a directcurrent voltage and radio frequency potential while a second set ofelectrodes applies an opposite direct current voltage and a radiofrequency potential that is phase shifted by 180 degrees with respect tothe radio frequency potential generated by the first set of electrodes.This radio frequency quadrupole holds a charged particle (e.g., nucleicacid) in the center of the field (i.e., center of the pore). Holding thetranslocating coded molecule in the middle of the nanopore is predictedto reduce the variability of electron flow through a pore and may alsoprovide consistency in current flow measured by electron tunneling. Itis suggested that altering the amplitude of the radio frequencyquadrupole could also be used to force the coded molecule to one side ofthe nanopore and slow the rate of translocation through the pore.

5.5 Signal Pattern Detection

Interrogating the coded molecule by translocation through a nanopore anddetecting the detectable property generates a signal pattern. Thecombination of the signals from each distinctive region of the codedpolymer (e.g., block polymer region, ligated ligation probe, etc.) formsa composite signal pattern that identifies the coded molecule. The typeof detection method employed will correspond to the property beingdetected for the polymers that make up the coded molecule.

In some embodiments, the detectable property is the effect of the codedmolecule on the electrical properties of the nanopore as the codedmolecule translocates through the pore. Electrical properties of thenanopore include among others, current amplitude, impedance, duration,and frequency. Devices for detecting the pore's electrical propertiestypically comprises a nanopore incorporated into a thin film or amembrane, where the film or membrane separates a cis chamber and a transchamber connected by a conducting bridge. The coded molecule to beanalyzed is placed on the cis side of the nanopore in an aqueoussolution typically comprising one or more dissolved salts, such aspotassium chloride. Application of an electric field across the poreusing electrodes positioned in the cis and trans side of the nanoporecauses translocation of the coded molecule through the nanopore, whichaffects the migration of ions through the pore, thereby altering thepore's electrical properties. Current is measured at a suitable timefrequency to obtain sufficient data points to detect a current signalpattern. The generated signal pattern can then be compared to a set ofreference patterns in which each reference pattern is obtained fromexamination of a single population of known coded molecules. Shifts incurrent amplitude, current duration, and current magnitude define asignal pattern for the coded molecule. Measurement of current propertiesof a nanopore, such as by patch clamp techniques, is described inpublications discussed above and in various reference works, forexample, Hille, B, 2001, Ion Channels of Excitable Membranes, 3rd Ed.,Sinauer Associates, Inc., Sunderland, Mass.

In some embodiments, the detectable property of the coded molecule isquantum tunneling of electrons. Quantum tunneling is thequantum-mechanical effect of transitioning through aclassically-forbidden energy state via a particle's quantum waveproperties. Electron tunneling occurs where a potential barrier existsfor movement of electrons between a donor and an acceptor. To detectelectron tunneling, a microfabricated electrode tip is positioned about2 nanometers from the specimen. At an appropriate separation distance,electrons tunnel through the region between the tip and the sample, andif a voltage is applied between the tip and the sample, a net current ofelectrons (i.e., tunneling current) flows through the gap in thedirection of the voltage bias. Where the nanodevice uses detectionelectrodes for measuring tunneling current, the electrodes arepositioned proximately to the translocating coded molecule such thatthere is electron tunneling between the detection electrodes and codedmolecule. As further discussed below, the arrangement of the electrodesrelative to the translocating coded molecule will dictate the type ofelectron transport occurring through the coded molecule.

In some embodiments, analysis of the coded molecule involves detectingcurrent flow occurring through the nucleic acid chain (i.e.,longitudinally along the nucleic acid chain) (Murphy et al., 1994, ProcNatl Acad Sci USA 91(12):5315-9). The exact mechanism of electrontransfer is unknown, although electron tunneling is given as oneexplanation for DNA's transport properties. However, the physicsunderlying electron transport through a double-stranded nucleic acid isnot limiting for the purposes herein, and detection of current flowingthrough the nucleic acid serves to distinguish one polymer region of thecoded molecule from another polymer region, and hence distinguish onecoded molecule from another coded molecule. For detection of electronelectron flow occurring longitudinally through the coded molecule chain,the detection electrodes are positioned longitudinally to the directionof coded molecule translocation such that there is a gap between theelectrodes parallel to the chain of an extended coded molecule. Invarious embodiments, the detection electrodes may be placed on oppositesides of a layer(s) (e.g., membrane) separating the two sides of thenanopore, while in other embodiments, the detection electrodes may bepositioned within the layer(s) that separate the two sides of thenanopore.

Another mode of electron flow in a nucleic acid is that occurring acrossthe nucleic acid, for example, a direction transverse to an extendednucleic acid chain (e.g., across the diameter of a double-strandednucleic acid). In a double-stranded nucleic acid, electron transport mayoccur through the paired bases while in a single-stranded nucleic acid,electron transport may occur through a single unpaired base.Furthermore, differences in the chemical compositions, hydrationstructures, interactions with charged ions, spatial orientation of eachbase, and different base pairing combinations will alter the transverseelectron transport characteristics, and thus provide a basis fordistinguishing a coded molecule that differ in sequence and/or polymerbackbone. For detection of electron flow across the coded molecule(i.e., transverse to an extended nucleic acid chain), the detectionelectrodes are positioned on one side of the nanopore to interrogate thecoded molecule across rather than through the nanopore.

In embodiments of longitudinal or transverse detection, the thickness ofthe electrodes may determine the total number of bases interrogated bythe electrodes. For transverse detection, the tips of the detectionelectrodes may be dimensioned to interrogate a single nucleobase, andthereby obtain single base resolution. In other embodiments, thedimensions of the detection electrode are arranged to interrogate morethan one nucleobase. Thus, in some embodiments, the number ofnucleobases interrogated at any one time may be about 2 or more, about 5or more, about 10 or more, or about 20 or more depending on theresolution required to detect differences in the various polymer regionsof the coded molecule.

In some embodiments, the coded molecule is detected using an electrontunneling probe, such as that used in an electron tunneling microscope.In these embodiments, the electrode tip is rastered across a smallregion of the sample. As the tip scans the surface, differences in theelectron density at the surface of the sample cause correspondingvariations in the tunneling current. Changes in tunneling currentprovide a map of the variations in electron density at the surface ofthe coded molecule. For the embodiments herein, the coded molecule maybe absorbed onto a surface in an extended conformation and then scannedusing an electrode tip. In other embodiments, the electrode tip of theelectron tunneling microscope is held stationary while the codedmolecule is translocated across the tip. A device for translocating anucleic acid across an electrode probe is described in PCT publicationWO 00/79257.

In other embodiments, differences in the structure and spatialorientation of a coded molecule may be detected as differences incapacitance. This type of measurement is illustrated in U.S. applicationpublication no. 2003/0141189. Capacitance causes a phase shift in anapplied ac voltage at a defined applied frequency and impedance. Phaseshift characteristics for each nucleobase is determined for nucleicacids of known sequence and structure, and used as reference standardsfor identifying individual base characteristics. Nearest neighboranalysis may permit capacitance measurements extending to more than asingle nucleobase.

In other embodiments, the detection technique is based on imagingcharge-induced fields, as described in U.S. Pat. No. 6,413,792 and U.S.published application No. 2003/0211502, the disclosures of which areincorporated herein by reference. For detecting coded molecules based oncharge induced fields, a semiconductor device described above is used.Application of a voltage between a source region and a drain regionresults in flow of current from the source to the drain if a channel forcurrent flow forms in the semi-conductor. Because each nucleobase has anassociated charge, passage of a coded molecule through the semiconductorpore induces a change in the conductivity of the semiconductor materiallining the pore, thereby inducing a current of a specified magnitude andwaveform. Currents of differing magnitude and waveform are produced bydifferent bases because of differences in charge, charge distribution,and size of the bases. In the embodiments disclosed in U.S. Pat. No.6,413,792, the polymer passes through a pore formed of a p-type siliconlayer. Translocation of the coded molecule is achieved by methodssimilar to those used to move a polymer through other types of channels,as described above. The magnitudes of the current is expected to be onthe order of microampere range, which is much higher than the expectedpicoampere currents detected by electron tunneling. Because the polymerblock regions in the coded molecule comprise more than a singlenucleobase, these block polymer regions should produce distinctivesignals reflective of the charge and charge distribution of the blockpolymer regions.

It is to be understood that although descriptions above relate toindividual detection techniques, in some embodiments, a plurality ofdifferent techniques may be used to examine a single coded molecule(see, e.g., Kassies et al., 2005, J Microsc 217:109-16). Examples ofmultiple detection modes include, among others, current blockade incombination with electron tunneling current, and current blockage incombination with imaging charge induced fields. Concurrent detectionwith different detection modes may be used to identity a coded moleculeby correlating the detection time of the resulting signal betweendifferent detection modes.

5.6 Detection of Target Polynucleotides

In various embodiments, the target polynucleotide refers to thepolynucleotide detected by the coded molecule. The target polynucleotidecan be any nucleobase sequence, including but not limited to, genomicDNA (gDNA), RNA (e.g., mRNA; noncoding RNA, tRNA, siRNA, snRNA), nucleicacid obtained from subcellular organelles (e.g., mitochondria orchloroplasts), and nucleic acid obtained from microorganisms, parasites,or viruses. Furthermore, a target polynucleotide can be present insingle-stranded forms, multi-stranded forms (e.g., double-stranded,triple stranded), or a mixture of single-stranded and multi-strandedforms. The target polynucleotides can be linear, circular, or branched.

In some embodiments, the target polynucleotide can be an amplicongenerated by any suitable amplification technique including, but notlimited to polymerase chain reaction, oligonucleotide ligation assay,ligase chain reaction, reverse transcriptase PCR, invasive cleavage,rolling circle amplification, and strand displacement cleavage reactions(see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188;5,075,216; 5,130,238; 5,176,995; 5,185,243; 5,354,668; 5,386,022;5,427,930; 5,455,166; 5,516,663; 5,656,493; 5,679,524; 5,686,272;5,869,252 6,025,139; 6,040,166; 6,197,563; 6,297,016; 6,514,736; andEuropean Patent Nos. EP0200362, EP0201184, and EP320308). Ampliconssuitable for use in the methods and compositions described herein can beobtained from cells, cell lysates, and tissue lysates.

In various embodiments, the samples to be analyzed may be obtained fromvarious sources. “Sample” is to be used in the broad sense and isintended to include a wide range of environmental sources and biologicalmaterials, including compositions derived or extracted from suchbiological materials, such as amplicons described above. Non-limitingexamples of environmental samples include food, water, soil, waste, orair. Exemplary biological samples include, among others, whole blood;red blood cells; white blood cells; buffy coat; hair; nails and cuticlematerial; swabs (e.g., buccal swabs, throat swabs, vaginal swabs,urethral swabs, cervical swabs, throat swabs, rectal swabs, lesionswabs, abcess swabs, nasopharyngeal swabs, and the like); urine; sputum;saliva; semen; lymphatic fluid; amniotic fluid; cerebrospinal fluid;peritoneal effusions; pleural effusions; fluid from cysts; synovialfluid; vitreous humor; aqueous humor; bursa fluid; eye washes; eyeaspirates; plasma; serum; pulmonary lavage; lung aspirates; and tissues,including but not limited to, liver, spleen, kidney, lung, intestine,brain, heart, muscle, pancreas, biopsy material, and the like. Tissueculture cells, including explanted material, primary cells, secondarycell lines, and the like, as well as lysates, extracts, or materialsobtained from any cells, are also within the meaning of the termbiological sample as used herein. Microorganisms and viruses that may bepresent on or in a sample are also within the scope of the invention.Materials obtained from forensic settings are also within the intendedscope of the term sample.

The samples can be used without further processing or processedaccording to various methods typically used to prepare samples. Forinstance, samples containing cells or bacteria may be subjected tophysical conditions to disrupt the cells and liberate their contents.Non-limiting examples of such techniques include, among others,sonication, pressure, heat, irradiation, and mechanical shearing.Samples may also be treated with detergents, denaturing agents (e.g.,guanidinium chloride), chaotropic salts, and enzymes such as lysozymes,nucleases, glycosidases, etc. Samples may be subjected to furthermanipulation, such as filtration, chromatography, precipitation, solventextraction, and derivatization.

For detecting the target polynucleotide, the sample is contacted withthe coded molecule under conditions suitable for interaction of thetarget polynucleotide and the binding moiety. Typically, conditions arechosen that minimize non-specific interactions and stabilize annealingbetween the complementary regions of the target polynucleotide and thetarget probe. The conditions will vary depending on the type ofpolynucleotide and may be readily determined by the skilled artisan.Guidance is provided in various reference works, such as Sambrook etal., Molecule Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2001), Current Protocols in Molecular Biology,Ausubel. F. ed., Greene Pub. Associates (1998) updates to 2005; allpublications incorporated herein by reference. Factors for considerationinclude, among others, incubation time, pH, ionic strength, temperature,and divalent ion concentration. For nucleic acid detection, theseconditions can be varied to create a level of hybridization stringencythat minimizes hybridization of non-complementary sequences while beingstable to complementary target sequences.

In some embodiments, annealing characteristics of a nucleobase polymercan be determined by the T_(m) of the hybrid complex. The greater theT_(m) value, the more stable the hybrid. T_(m) is the temperature atwhich 50% of a nucleobase oligomer and its perfect complement form adouble-stranded oligomer structure. The T_(m) for a selected nucleobasepolymer also varies with factors that influence or affect hybridization.For example, such factors include, but are not limited to, factorscommonly used to impose or control stringency of hybridization, (i.e.,formamide concentration (or other chemical denaturant reagent), saltconcentration (i.e., ionic strength), hybridization temperature,detergent concentration, pH, and the presence or absence of chaotropes.Optimal stringency for forming a hybrid combination can be found by thewell-known technique of fixing several of the aforementioned stringencyfactors and then determining the effect of varying a single stringencyfactor. The same stringency factors can be modulated to control thestringency of hybridization of a PNA to a polynucleotide, except thatthe hybridization of a PNA is fairly independent of ionic strength.Optimal or suitable stringency for an assay can be experimentallydetermined by examination of each stringency factor until the desireddegree of discrimination is achieved.

The T_(m) values for the nucleobase oligomers can be calculated usingknown methods for predicting melting temperatures (see, e.g., Baldino etal., Methods Enzymology 168:761-777; Bolton et al., 1962, Proc. Natl.Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc. Natl. Acad. SciUSA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad. Sci USA83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychlik et al.,1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, Nucleic Acids Res19:698); Sambrook et al., supra); Suggs et al., 1981, In DevelopmentalBiology Using Purified Genes (Brown et al., eds.), pp. 683-693, AcademicPress; and Wetmur, 1991, Crit Rev Biochem Mol Biol 26:227-259. Allpublications incorporate herein by reference.

Following hybridization of the target probe to the target probe, thecoded molecule and target polynucleotide mixture is treated with amodifying agent as described above for the various embodiments.Conditions for the modification reactions can be standard conditions inwhich the modifying agents are known to be active. In some embodiments,the coded molecule can be isolated from unmodified molecules, such asthrough use of capture tags. In addition, prior to translocation of thecoded molecule through the nanopore, the coded molecule in someembodiments can be made single-stranded by any of known techniques(e.g., heating, chemical denaturation, etc.) to permit analysis insingle-stranded coded molecules.

For detecting a signal pattern, a coded molecule is placed into ananopore device and then driven or transported into the nanopore using asuitable force, typically a biased electric field. The driving force maybe constant or varied in a controlled manner, such as by use of pulsedcurrent. As the coded molecule translocates through the nanopore, thepolymer is interrogated to sense the detectable property of the codedmolecule. Interrogation of the coded molecule occurs sequentially as itis translocated through the nanopore. By “sensing” or “scanning” refersto the process of evaluating and/or interrogating the detectableproperty of the coded molecule in an orderly manner. The orientation ofthe coded molecule may be determined, relative to a reference ororientation point, for example but not limited to, a block polymerregion, a detectable tag, or a distinguishable sub-pattern of the signalpattern generated by the code molecule. The signal pattern can then becompared to a reference set of signal patterns to identity the codedmolecule sampled and relate or associate its identity to thecorresponding target probe and thus the target polynucleotide detected.In some embodiments, each coded molecule identified can be counted toquantitate the amount of a particular target polynucleotide present in asample.

In various embodiments, the form of the signal pattern depends on whichend of the coded molecule enters the nanopore. In order to decode andidentify the target probe in a mixture of coded molecule-labeled targetprobes, the signal pattern of each coded molecule can be unique. Thisorientation-dependent signal pattern can be addressed in various ways.In some embodiments, the signal patterns of a coded molecule enteringthe pore in both orientations can be obtained and decoded to associateit with a specific coded molecule. Although this method of associating aparticular set of signal patterns to a coded molecule reduces the numberof coded molecules that can be devised from a defined set of blockpolymer regions, the reduction in the number of usable coded moleculescan be compensated by simply increasing the number of block polymerregions used in the coded molecule. Block polymer combinations that areexpected to produce similar signal patterns in either orientation can beeliminated.

In other embodiments, the orientation of the coded molecule interrogatedthrough the nanopore can be assessed by determining the change in signalpattern of the coded molecule modified by the modifying agent. Themodification and corresponding change in signal pattern of the codedmolecule effectively serves as a marker for orientation of the codedmolecule in the nanopore. By using a modification that results in aunique signal, different from the signal pattern generated when thecoded molecule enters the pore through the other orientation, theorientation of the coded molecule can be readily determined. Forexample, the modification can be ligation probes with different signalgenerating segments and/or ligation probes of differing nucleotidelengths, which generates an asymmetry in the signal pattern.

As noted above, the coded molecules can be used to detect a variety oftarget polynucleotides, including, among others, genomic DNA (gDNA); RNA(e.g., mRNA; noncoding RNA, tRNA, siRNA, snRNA); mitochondria orchloroplast DNA; nucleic acid obtained from microorganisms (e.g., fungi,bacteria); parasites (e.g., trypanosomes, nematodes, helminthes); DNA orRNA viruses; and synthetic nucleobase sequences (e.g., sequences forisolating a PCR product). In some embodiments, the target polynucleotidecan be from a pathogenic organism, non-limiting examples of whichinclude, among others, Salmonella, Campylobacter, Vibrio cholerae,Leishmania, enteric E. coli, retroviruses, herpesviruses, adenoviruses,and lentiviruses. In still other embodiments, the polynucleotide probesequences are directed to variants of a specific pathogen. For instance,drug resistant human immunodeficiency virus (HIV) can arise frommutations in the genes that encode the molecules targeted byanti-retroviral drugs, such as mutations in HIV gene encoding theprotease enzyme that renders the protease resistance to proteaseinhibitors used for HIV therapy. Thus, polynucleotide probe sequencesthat distinguish the various mutations can be used in the codedmolecules to detect protease resistant viral strains.

In some embodiments, the target polynucleotides detected are mutatedsequences associated with inherited disorders. Non-limiting examplesinclude, among others, mutations responsible for cystic fibrosis,hereditary nonpolyposis colorectal cancer, hemophilia, Huntington'sdisease, leukodystrophy, and sickle cell disease. Different mutationscausing each genetic disorder can be detected by use of a pair of codedmolecules for each mutation site, where one coded molecule has a targetprobe for the normal sequence and another coded molecule has a targetprobe for the mutated sequence.

In still other embodiments, the target polynucleotide detected isassociated with a sequence variation within a population. These sequencevariations have uses in evolutionary studies, familial relationshipanalysis, forensic analysis, disease diagnosis, disease prognosis, anddisease risk. As used herein, a “polymorphism” is a variation in the DNAsequence in some members of a species. A polymorphism is “allelic,” inthat, due to the existence of the polymorphism, some members of aspecies may have the unmutated sequence (i.e., the wild type “allele”)whereas other members may have a mutated sequence (i.e., the variant ormutant “allele”). When only one mutated sequence exists, thepolymorphism is referred to as “diallelic.” In the case of diallelicdiploid organisms, three genotypes are possible. The organism can behomozygous for one allele, homozygous for the other allele, orheterozygous. In the case of diallelic haploid organisms, they can haveone allele or the other, thus only two genotypes are possible. Theoccurrence of alternative mutations can give rise to trialleleic, etc.polymorphisms. Allelic polymorphisms referred to as “single nucleotidepolymorphisms,” or “SNPs” are polymorphism that contains a polymorphicsite, “X,” which is the site of the polymorphism's variation.

SNPs have several advantages for genotyping. SNPs are more stable thanother classes of polymorphisms, and SNPs occur at greater frequency andwith greater uniformity over a genetic region, which permits the use ofSNPs with tighter linkage to a particular phenotypic trait of interest.An exemplary SNP variation suited for the methods herein are thesequence variation associated with apolipoprotein E (ApoE), which iscorrelated with an increased risk for Alzheimer's Disease. The ApoE genedisplays polymorphisms predominantly at two nucleotide positions thatresult in three possible alleles for this gene: E2, E3, and E4. Eachallele, differing by one base, produces a protein product that differsby one or two amino acids from the other alleles. An individualinheriting at least one e4 allele has an increased risk of developingAlzheimer's while inheriting the E2 allele is not associated with anincreased risk.

Another example of useful SNP variations are those associate withcytochrome P450 enzymes, a superfamily of heme containingmonooxygenases. Human cytochrome P450 enzyme families, such as CYP1,CYP2, and CYP3, metabolize various drugs and environmental chemicalssuch that differences in the activities of specific enzymes within eachcytochrome P450 family can affect drug metabolism (Gonzalez, F. J.,1992, Trends Pharmacol Sci 13(9)346-52). An SNP that results in low orno expression of CYP2C9 can increase the risk of adverse effects oftaking tolbutamide or coumadin because of the low metabolism of thesedrugs in subjects carrying the SNP in the CYP2C9 (Schwarz, U. I., 2003,Eur J Clin Invest 33 Suppl 2:23-30). Detecting polymorphisms in theseand other drug metabolizing enzymes (e.g., esterases) can be used topredict a subject's response to a drug.

As will be apparent to the skilled artisan based on the guidance herein,uses of the coded molecule in methods above are numerous, and areapplicable to detection of target nucleotides other than thosespecifically described. Furthermore, the multiplexing capabilities ofthe methods allow its use to detect a large number of different targetpolynucleotides in a single assay.

5.7 Kits

The coded molecules and devices for their analysis can be provided inthe form of kits. The kits can comprise coded molecules for detectingtarget polynucleotides and corresponding coded molecule referencestandards, including unmodified coded molecules and modified codedmolecules for comparing signal patterns of test samples. Kits canfurther include nanopore devices created on a single chip for detectingthe coded molecules. In various embodiments, the kits can also includeinstructions for proper use of the coded molecules and nanopore devices.Instructions and diagrams can be on any medium, non limiting examples ofwhich include, printed forms, magnetic tape, flash memory, compact disc,and magnetic disks.

The foregoing descriptions of embodiments have been presented forpurposes of illustration and description and are not intended to beexhaustive or to limit the scope of the disclosure to the precise formsdisclosed. The teachings herein arc intended to encompass variousalternatives, modifications, and equivalents, as will be appreciated bythose of skill in the art.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1.-73. (canceled)
 74. A method of detecting a target polynucleotide,comprising: a) contacting a coded molecule with a target polynucleotide,wherein the coded molecule comprises (i) one or more block polymerregions, and (ii) a target probe capable of hybridizing to the targetpolynucleotide; b) modifying the target probe with a modifying agent,wherein the modification is dependent on the target polynucleotidehybridized to the target probe; and c) translocating the coded moleculethrough a nanopore and detecting a signal pattern associated with thecoded molecule.
 75. The method of claim 74, further comprising the stepof rendering the coded molecule single-stranded prior to translocationthrough the nanopore.
 76. The method of claim 74, further comprisingcomparing the detected signal pattern to a signal pattern of anunmodified coded molecule, wherein a difference in the detected signalpattern of the modified coded molecule compared to the signal pattern ofthe unmodified coded molecule indicates the presence of the targetpolynucleotide.
 77. The method of claim 74, further comprisingassociating the detected signal pattern to the target probe.
 78. Themethod of claim 74, in which the target polynucleotide comprises a5-prime region and a 3-prime region, and the target probe comprises a3-prime terminal sequence that hybridizes to the 5-prime region of thetarget polynucleotide, and wherein the modifying agent is atemplate-dependent polymerase and the modification is extension of thehybridized 3-prime region of the target probe.
 79. The method of claim78 in which the 3-prime terminal sequence of the target probe comprisesa 3-prime terminal nucleotide that interrogates a site of nucleotidepolymorphism on the target polynucleotide.
 80. The method of claim 78 inwhich the target polynucleotide comprises a circular nucleic acid,wherein the circular nucleic acid is a ligated open circle probe (OCP).90. The method of claim 80 in which the ligated OCP is formed byhybridization of the OCP terminal regions to a nucleic acid of interest.91. The method of claim 78 in which the primer extension is carried outin the presence of one or more nucleotide triphosphates.
 92. The methodof claim 91 in which all four of the nucleotide triphosphates arepresent.
 93. The method of claim 74 in which the target polynucleotidecomprises adjacent first and second regions, and the method furthercomprises hybridizing a ligation probe to the target polynucleotide,wherein the ligation probe hybridizes to the first region and the targetprobe hybridizes to the second region of the target polynucleotide suchthat a terminus of the ligation probe and a terminus of the target probeare adjacent, and wherein the modifying agent is a ligase and themodification is ligation of the ligation probe to the target probe. 94.The method of claim 93 in which the ligase is a DNA ligase.
 95. Themethod of claim 94 in which the terminus of the target probe comprises aterminal nucleotide that interrogates a site of nucleotide polymorphismon the target polynucleotide.
 96. The method of claim 95 in which thetarget probe terminal nucleotide is a 5-prime or 3-prime terminalnucleotide.
 97. The method of claim 94 in which the terminus of theligation probe comprises a terminal nucleotide that interrogates a siteof nucleotide polymorphism on the target polynucleotide.
 98. The methodof claim 97 in which the ligation probe terminal nucleotide is a 5-primeor 3-prime terminal nucleotide.
 99. The method of claim 94 in which theligation probe further comprises a signal generating segment.
 100. Themethod of claim 74 in which the target polynucleotide comprises adjacentfirst and second regions, and the target probe comprises a 5-primeregion and a 3-prime region, and wherein the method further compriseshybridizing a FLAP probe to the target polynucleotide, wherein the FLAPprobe comprises a 3-prime segment that hybridizes to the first region,and the 3-prime region of the target probe hybridizes to the secondregion such that the 3-prime segment of the FLAP probe and the 3-primeregion of the target probe are adjacently hybridized to the targetpolynucleotide to form a FLAP substrate, and wherein the modifying agentis a FLAP endonuclease and the modification is cleavage of the targetprobe.
 101. The method of claim 96 in which the 5-prime region of thetarget probe is non-complementary to the target polynucleotide.
 102. Themethod of claim 96 in which the FLAP probe further comprises a 3-primeunpaired segment that overlaps with the 5-prime region of the targetprobe in the FLAP substrate, thereby forming a double FLAP substrate.